Artificial protein cages with guest cargo encapsulated within them

Abstract

The present invention provides an artificial TRAP cage that includes a selected number of TRAP rings and a guest cargo encapsulated therein.

Description

[Technical field]

[0001] The present invention is in the field of biochemistry.The present invention relates to an artificial protein cage, called a "TRAP cage", which contains a selected number of TRAP rings and a guest cargo encapsulated therein. [Background technology]

[0002] Proteins that associate into monodisperse cage-like structures are useful molecular containers for a variety of applications in biotechnology and medicine. Such protein cages exist in nature, for example in viral capsids, but can also be designed and constructed in the laboratory.

[0003] Therefore, we have previously described that TRAP-K35C, a single cysteine ​​mutant of tryptophan RNA-binding attenuator protein from Geobacillus stearothermophilus, can assemble into hollow sphere structures composed of multiple cyclic 11-mer subunits via reaction with gold nanoparticles. 1 The resulting protein cages are extremely stable under many harsh conditions but readily disassemble into capsomere units upon the addition of reducing agents.

[0004] Although these attractive features of the TRAP cage make it ideal for developing intracellular delivery vehicles, a fundamental challenge remains guest packaging. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide a simple and robust method for the internal loading of TRAP cages with proteins or therapeutic agents of interest in a stoichiometrically controllable manner. [Means for solving the problem]

[0006] The subject of the present invention is an artificial TRAP cage comprising a selected number of TRAP rings and at least one guest cargo encapsulated therein.Preferably, the artificial TRAP cage comprises a selected number of TRAP rings held in place by a crosslinker.Preferably, the crosslinker is a molecular crosslinker or an atomic metal crosslinker.Preferably, the TRAP rings are linked by gold or DTME.

[0007] Preferably, the guest cargo is equal to or smaller than the diameter of the TRAP cage lumen. Preferably, the guest cargo is less than 16 nm in diameter. Preferably, the guest cargo is between 4 nm and 16 nm in diameter. Guest cargo larger than 4 nm will not be able to diffuse into or out of the TRAP cage.

[0008] Preferably, the cargo is a protein, preferably selected from the group comprising enzymes (e.g., proteases, nucleases, hydrogenases, dehydrogenases, lipases, lyases, ligases, transferases, reductases, recombinases, nucleases acid modifying enzymes, or other types of enzymes), antigens, antibodies. Alternatively, the cargo is another type of protein biological macromolecule (e.g., sterols, steroids, or fatty acids). Alternatively, the cargo is a lipid, a peptide (e.g., peptide hormones, cell membrane disrupting peptides, T cell stimulating peptides, or another type of peptide), a nucleic acid (e.g., DNA, engineered DNA nanostructures, including those designed using DNA origami techniques, DNAzymes, RNA, mRNA, miRNA, siRNA, tRNA, single stranded RNA, double stranded RNA, RNAzymes), small molecule cargo such as drugs, peptide nucleic acids (PNAs), carbon-based structures (e.g., fullerenes or buckminsterfullerenes, single-walled or multi-walled carbon nanotubes). ), metals (e.g., iron, zinc, platinum, copper, sodium, cadmium, lanthanides, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof), toxins (e.g., ligand-targeted toxins, protease-activating toxins, melittin, and toxin-based suicide gene therapy drugs), or nanoparticles (e.g., metal nanoparticles such as gold, iron, silver, cobalt-cadmium-selenium, titanium dioxide, etc.) or core-shell metal nanoparticles such as CdS / ZnS, CdSe / ZnS, CdSe / CdS, and InAs / CdSe nanoparticles.

[0009] Preferably, the nucleic acid is selected from the group comprising DNA, RNA, mRNA, siRNA, tRNA, and microRNA.

[0010] Preferably, the therapeutic agent is an enzyme associated with overexpression in a metabolic disorder or disease, or underexpression in a metabolic disorder or disease.

[0011] Preferably, the enzyme is selected from the group comprising hydrogenases, dehydrogenases, lipases, lyases, ligases, proteases, transferases, reductases, recombinases, and nucleases acid modifying enzymes.

[0012] Preferably, the therapeutic agent is selected from the group including cancer therapeutics, anti-infective therapeutics, vascular disease therapeutics, immunotherapeutics, senolytics, and neurological therapeutics.

[0013] Preferably, the metal is selected from the group including iron, zinc, platinum, copper, sodium, cadmium, lanthanides, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof.

[0014] Preferably, the toxin is selected from the group including ligand-targeted toxins, protease-activated toxins, melittin, and toxin-based suicide gene therapy drugs.

[0015] Preferably, the guest cargo is a protein. Preferably, a fluorescent protein. Preferably, GFP, mCherry, or mOrange. Preferably, Interleukin-2 (IL-2) or Neoleukin-2 / 15 (NL-2).

[0016] Preferably, the therapeutic agent is selected from the group including cancer therapeutics, anti-infective therapeutics, vascular disease therapeutics, immunotherapeutics, senolytics, and neurological therapeutics.

[0017] Preferably, the cage comprises a plurality of cargoes, preferably the cargoes being the same or different from one another.

[0018] Preferably, the TRAP cage according to the present invention further comprises at least one external decoration.

[0019] Preferably, at least one of the external decorations comprises a cell permeation agent that facilitates intracellular delivery of the cage containing the internal guest cargo.

[0020] Preferably, the cell permeability agent is PTD4.

[0021] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0022] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0023] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0024] Preferably, the inner surface of the TRAP cage lumen is supercharged. Preferably, the TRAP cage with a supercharged lumen comprises an E48Q or E48K mutation. Preferably, the TRAP cage with a supercharged lumen comprises a K35C / E48Q or K35C / E48K mutation.

[0025] Preferably, the guest cargo is genetically fused to the inner surface of the TRAP cage lumen. Preferably, the genetic fusion of the guest cargo to the inner surface of the TRAP cage lumen in step (ii) is performed by genetically fusion of the guest cargo to the TRAP cage lumen facing the inner surface of the lumen. K35Cvia N-terminal fusion of a guest cargo to the N-terminus of the. Preferably, the guest cargo is a genetic fusion protein, preferably a fluorescent protein, preferably GFP, mCherry, or mOrange. Preferably, the genetic fusion protein is interleukin-2 (IL-2) or neoleukin-2 / 15 (NL-2).

[0026] Preferably, the guest cargo is conjugated, preferably to the inner surface of the TRAP cage lumen, using SpyCatcher / SpyTag conjugation. Preferably, in the SpyCatcher / SpyTag conjugation of the guest cargo to the inner surface of the TRAP cage lumen in step (iii), SpyCatcher is introduced into the loop region of the TRAP ring between residues 47 and 48 that faces inward when associated with the TRAP cage, and the guest cargo contains a SpyTag.

[0027] Preferably, the guest cargo is attached to the TRAP cage, preferably to the inside of the TRAP cage, via a covalent bond, preferably by chemical or enzymatic bond formation.

[0028] Preferably, the opening of the cage is programmable.Preferably, said specific conditions correspond to specific cleavage characteristics of the cross-linker.

[0029] Preferably, the programmable opening of the cage relies on the selection of a molecular or atomic metal crosslinker that holds the TRAP ring in place in the TRAP cage.

[0030] Preferably, the specific cleavage characteristics of the molecular crosslinker are: (i) reduction-resistant / insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions; (ii) reduction-responsive / sensitive molecular crosslinkers, whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular crosslinker, whereby the cage opens upon exposure to light The compound is selected from the group consisting of:

[0031] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimideohexane (BMH) and bis-bromoxylene. Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimideoethane (DTME). Preferably, the photoactivatable molecular crosslinker may be selected from the group including bis-halomethylbenzene and its derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0032] Preferably, the molecular cross-linker is a homobifunctional molecular moiety and its derivatives. Preferably, the homobifunctional molecular cross-linker is bismaleimidohexane (BMH).

[0033] Preferably, the cage is resistant / insensitive to reducing conditions.Preferably, the homobifunctional molecular crosslinker is dithiobismaleimideethane (DTME).

[0034] Preferably, the cage is responsive / sensitive to reducing conditions.Preferably, the molecular cross-linker is a bis-halomethylbenzene and its derivatives.

[0035] Preferably, the molecular crosslinker is selected from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0036] Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

[0037] Preferably, a cage according to the present invention comprises a mixture of different programmable molecular cross-linkers.

[0038] Preferably, the TRAP ring is a variant.

[0039] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, E48Q, E48K, R64S, K35C / E48Q, K35C / E48K, and K35C / R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation or a K35C / E48Q mutation or a K35C / E48K mutation.

[0040] Preferably, the artificial TRAP cage protein is selected from the group consisting of K35C, K35H, R64S, E48Q, E48K, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, S33H / K35C, K35C / E48Q, K35C / E48K, K35H / E48Q, K35H / E48K, S33C / E48Q, S33C / E48K, S33C / E48Q, S The artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including 33C / R64S / E48Q, K35H / S33C / R64S, S33C / R64S, S33H / K35H / R64S, S33C / K35C / R64S, K35C / R64S / E48Q, K35H / R64S / E48Q, K35H / R64S / E48K, S33C / R64S / E48Q, S33C / R64S / E48K, S33C / R64S / E48Q, and S33C / E48K. Preferably, the artificial TRAP cage protein is modified to include K35H / E48Q or K35H / E48 mutations.

[0041] Preferably, the TRAP cage is stable at elevated temperatures, i.e. when the temperature is elevated above normal room temperature or human / animal body temperature, preferably stable between 0 and 100°C, preferably stable between 15 and 100°C, preferably stable between 15 and 79°C, preferably stable up to 95°C, preferably stable below 95°C.

[0042] Preferably, the TRAP cage is stable at non-neutral pH, preferably above pH 7 and below pH 7, preferably stable at pH 3-11, preferably stable at pH 4-10, preferably stable at pH 5-9.

[0043] Preferably, the TRAP cage is stable in chaotropic agents (agents that disrupt hydrogen bonds in solution, that would disrupt or denature protein or macromolecular structures) or detergents that would otherwise be expected to disrupt or denature protein or macromolecular structures. Preferably, the cage exhibits stability in n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. Preferably, the TRAP cage is stable in up to 4M GndHCl. Preferably, the TRAP cage is stable in up to at least 7M urea. Preferably, the TRAP cage is stable in up to 15% SDS. The stability of the cages described herein may be tested using these agents in standard conditions that would be known to those skilled in the art to demonstrate said stability.

[0044] The cages described herein exhibit unexpected stability in these conditions, providing a more stable TRAP cage than previously demonstrated.

[0045] A subject of the present invention is also the use of a cage according to the invention as defined above in the delivery of a cargo in a controlled period of time and to a desired location.

[0046] A subject of the present invention is also the use of an artificial TRAP cage according to the invention as a delivery vehicle for the intracellular delivery of its internal guest cargo.

[0047] A subject of the present invention is also the use of an artificial TRAP cage according to the invention as a vaccine.

[0048] A subject of the present invention is also the use of an artificial TRAP cage according to the present invention for the treatment of a disease or pathological condition selected from the group comprising cancer, vascular diseases, cardiovascular diseases, diabetes, infectious diseases, autoimmune conditions, neurodegenerative diseases, cellular aging diseases, arthritis and respiratory diseases.

[0049] The subject of the present invention is a method for making an artificial TRAP cage with an encapsulated guest cargo, the method comprising: (i) obtaining a TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system; (ii) conjugation of a TRAP ring unit via at least one free thiol linkage with a crosslinker; (iii) modification of the TRAP ring unit to provide a suitable internal environment for capturing guest cargo; (iv) self-association to form a TRAP cage to provide a cage lumen in which guest cargo is encapsulated; and (v) Purification and isolation of TRAP cages encapsulating guest cargo The present invention is also a method including the steps of:

[0050] Preferably, step (ii) first comprises the conjugation of the TRAP ring unit via at least one metal crosslinker, preferably an atomic metal crosslinker. Step (ii) then comprises replacing the metal crosslinker with a molecular crosslinker. The molecular crosslinker can exchange the metal atom without changing the orientation of the ring in the cage. Preferably, the metal is gold. This modified step (ii) is preferably applied when the crosslinker is a photocleavable linker, preferably when the crosslinker is bromoxyl or bisbromobimane.

[0051] Preferably, the modification in step (iii) is (i) highly charging the inner surface of the TRAP cage lumen; (ii) genetic fusion of guest cargo to the inner surface of the TRAP cage lumen; (iii) SpyCatcher / SpyTag conjugation of guest cargo to the inner surface of the TRAP cage lumen; and (iv) via covalent bond formation, both chemical and enzymatically The compound is selected from the group consisting of:

[0052] Preferably, the highly charging of the inner surface in step (i) provides either a net positive or a net negative charge on the inner surface of the cage lumen.

[0053] Preferably, the cargo is a protein, preferably selected from the group comprising enzymes (e.g., proteases, nucleases, hydrogenases, dehydrogenases, lipases, lyases, ligases, transferases, reductases, recombinases, nucleases acid modifying enzymes, or other types of enzymes), antigens, antibodies. Alternatively, the cargo is another type of protein biological macromolecule (e.g., sterols, steroids, or fatty acids). Alternatively, the cargo is a lipid, a peptide (e.g., peptide hormones, cell membrane disrupting peptides, T cell stimulating peptides, or another type of peptide), a nucleic acid (e.g., DNA, engineered DNA nanostructures, including those designed using DNA origami techniques, DNAzymes, RNA, mRNA, miRNA, siRNA, tRNA, single stranded RNA, double stranded RNA, RNAzymes), small molecule cargo such as drugs, peptide nucleic acids (PNAs), carbon-based structures (e.g., fullerenes or buckminsterfullerenes, single-walled or multi-walled carbon nanotubes). ), metals (e.g., iron, zinc, platinum, copper, sodium, cadmium, lanthanides, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof), toxins (e.g., ligand-targeted toxins, protease-activating toxins, melittin, and toxin-based suicide gene therapy drugs), or nanoparticles (e.g., metal nanoparticles such as gold, iron, silver, cobalt-cadmium-selenium, titanium dioxide, etc.) or core-shell metal nanoparticles such as CdS / ZnS, CdSe / ZnS, CdSe / CdS, and InAs / CdSe nanoparticles.

[0054] Preferably, the number of TRAP rings in the TRAP cage is 6-60, preferably 7-55, preferably 8-50, preferably 9-45, preferably 10-40, preferably 11-35, preferably 12-34, preferably 13-33, preferably 14-32, preferably 15-31, preferably 16-30, preferably 17-29, preferably 18-28, preferably 19-27, preferably 20-26. Preferably, the number of TRAP rings in the TRAP cage is less than 40, preferably less than 35, preferably less than 30. Preferably, the number of TRAP rings in the TRAP cage is more than 6, preferably more than 10, preferably more than 15, preferably more than 20.

[0055] Preferably, the number of TRAP rings in the TRAP cage is 12 to 24.

[0056] Preferably, the number of TRAP rings in the TRAP cage is about 24, preferably 24. Preferably, the number of TRAP rings in the TRAP cage is about 12, preferably 12. Preferably, the number of TRAP rings in the TRAP cage is about 20, preferably 20.

[0057] Preferably, the opening of the cage is programmable.Preferably, said specific conditions correspond to specific cleavage characteristics of the cross-linker.

[0058] Preferably, the programmable opening of the cage relies on the selection of a molecular or atomic metal crosslinker that holds the TRAP ring in place in the TRAP cage.

[0059] Preferably, the specific cleavage characteristics of the molecular crosslinker are: (i) reduction-resistant / insensitive molecular crosslinkers, whereby the cage remains closed under reducing conditions; (ii) reduction-responsive / sensitive molecular crosslinkers, whereby the cage opens under reducing conditions; and (iii) a photoactivatable molecular crosslinker, whereby the cage opens upon exposure to light The compound is selected from the group consisting of:

[0060] Preferably, the reduction-resistant / insensitive molecular crosslinker may be selected from the group including bismaleimidohexane (BMH) and bis-bromoxylene. Preferably, the reduction-responsive / sensitive molecular crosslinker may be selected from the group including dithiobismaleimidoethane (DTME). Preferably, the photoactivatable molecular crosslinker may be selected from the group including bis-halomethylbenzene and its derivatives including 1,2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0061] Preferably, the molecular cross-linker is a homobifunctional molecular moiety and its derivatives. Preferably, the homobifunctional molecular cross-linker is bismaleimidohexane (BMH).

[0062] Preferably, the cage is resistant / insensitive to reducing conditions.Preferably, the homobifunctional molecular crosslinker is dithiobismaleimideethane (DTME).

[0063] Preferably, the cage is responsive / sensitive to reducing conditions.Preferably, the molecular cross-linker is a bis-halomethylbenzene and its derivatives.

[0064] Preferably, the molecular crosslinker is selected from the group comprising 1,2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0065] Preferably, the molecular crosslinker is photolabile upon exposure to UV light.

[0066] Preferably, a cage according to the present invention comprises a mixture of different programmable molecular cross-linkers.

[0067] Preferably, the inner surface of the TRAP cage lumen is highly charged. Preferably, the TRAP cage with a highly charged lumen comprises an E48Q or E48K mutation. Preferably, the TRAP cage with a highly charged lumen comprises a K35C / E48Q or K35C / E48K mutation.

[0068] Preferably, the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including K35C, E48Q, E48K, R64S, K35C / E48Q, K35C / E48K, and K35C / R64S. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation. Preferably, the artificial TRAP cage protein is modified to include a K35C mutation or a K35C / E48Q mutation or a K35C / E48K mutation.

[0069] Preferably, the artificial TRAP cage protein is selected from the group consisting of K35C, K35H, R64S, E48Q, E48K, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, S33H / K35C, K35C / E48Q, K35C / E48K, K35H / E48Q, K35H / E48K, S33C / E48Q, S33C / E48K, S33C / E48Q, S The artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group including 33C / R64S / E48Q, K35H / S33C / R64S, S33C / R64S, S33H / K35H / R64S, S33C / K35C / R64S, K35C / R64S / E48Q, K35H / R64S / E48Q, K35H / R64S / E48K, S33C / R64S / E48Q, S33C / R64S / E48K, S33C / R64S / E48Q, and S33C / E48K. Preferably, the artificial TRAP cage protein is modified to include K35H / E48Q or K35H / E48 mutations.

[0070] Preferably, TRAP K35C E48QThe cage formation step of part (iii) for is carried out in sodium bicarbonate buffer at pH 9-11.

[0071] Preferably, TRAP K35C E48k The cage formation step of part (iii) for is carried out in sodium bicarbonate buffer at pH 10-10.5.

[0072] Preferably, the guest cargo can be loaded either before or after assembly of the TRAP cage.

[0073] Preferably, the genetic fusion of the guest cargo to the inner surface of the TRAP cage lumen in step (ii) is carried out by integrating the TRAP K35C via N-terminal fusion of a guest cargo to the N-terminus of the. Preferably, the guest cargo is a genetic fusion protein, preferably a fluorescent protein, preferably GFP, mCherry, or mOrange. Preferably, the genetic fusion protein is interleukin-2 (IL-2) or neoleukin-2 / 15 (NL-2).

[0074] Preferably, in step (iii) of SpyCatcher / SpyTag conjugation of a guest cargo to the inner surface of the TRAP cage lumen, SpyCatcher is introduced into the loop region of the TRAP ring between residues 47 and 48 that faces inward when associated with the TRAP cage, and the guest cargo contains a SpyTag.

[0075] Preferably, the enzymatic modification is mediated by a peptide ligase selected from the group including sortases, asparaginyl endoproteases, trypsin-related enzymes, and subtilisin-derived variants, and the covalent chemical bond formation may include strain promoted alkyne-azide cycloaddition and pseudopeptide bonds.

[0076] If cysteines are not present in the biomolecule, or they are present but not available for reaction, preferably an -SH group as the group of cysteine ​​can be introduced into the biomolecule.

[0077] The introduction of cysteine ​​can be carried out by any method known in the art.For example, the introduction of cysteine ​​can be carried out by methods known in the art, such as, but not limited to, commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers.The above-mentioned methods are known by those skilled in the art, and ready-to-use kits with protocols are commercially available.

[0078] The -SH moiety can also be introduced into a biomolecule by alteration of other amino acids in the biomolecule, ie, by site-directed mutagenesis or by solid phase peptide synthesis.

[0079] The subject of the present invention is also the TRAP cages produced by this method. These cages may have any of the characteristics or properties described in connection with the first aspect of the invention above, or any others described herein.

[0080] A subject of the present invention is also the use of a cage according to the invention as defined above in the delivery of a cargo in a controlled period of time and to a desired location.

[0081] A subject of the present invention is also the use of any of the TRAP cages described herein as a medicament.

[0082] A subject of the present invention is also the use of any of the TRAP cages described herein in treating a disease in a patient.

[0083] The subject of the present invention is also a method for treating a patient, comprising administering to said patient a TRAP cage as described herein.The subject of the present invention is also a method for treating an individual suffering from a disease selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infectious disease, autoimmune disease, neurodegenerative and neurological disease, cellular aging disease, arthritis, and respiratory disease, and in need of therapy, comprising administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargos selected from the group comprising nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies, and toxins, and fragments thereof of all the above that have therapeutic value.

[0084] The subject of the present invention is also a method for vaccinating an individual in need of vaccination due to a condition selected from the group including cancer, vascular diseases, cardiovascular diseases, diabetes, infectious diseases, autoimmune conditions, neurodegenerative and neurological diseases, cellular aging diseases, arthritis, and respiratory diseases, the method comprising the step of administering a therapeutically effective amount of an artificial TRAP cage with one or more internal guest cargos selected from the group including nucleic acids, enzymes, therapeutic agents, small molecules, organic or inorganic nanoparticles, peptides, metals, antigens, antibodies, and toxins, and fragments thereof of all the aforementioned that have therapeutic value.

[0085] Preferably, the TRAP caged therapeutic is administered via intranasal inhalation or injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] References herein to a "TRAP protein" refer to the bacterial protein tryptophan RNA-binding attenuating protein, which may be isolated, for example, from wild-type Geobacillus stearothermophilus or other such bacteria. Although the protein may be isolated from a variety of bacteria, TRAP proteins which will function as described herein include those from the following bacteria: Alkalihalobacillus ligniniphilus, Anaerobacillus isosaccharinicus, Anoxybacillus caldiproteolyticus, Anoxybacillus calidus, Anoxybacillus pushchinoensis, Anoxybacillus tepidamans, Anoxybacillus vitaminiphilus, Bacillaceae bacteria, Bacillus alveayuensis, Bacillus sinesaloumensis, Bacillus species FJAT-14578, Bacillus species HD4P25, Bacillus species HMF5848, Bacillus species PS06, Bacillus species REN16, Bacillus species SA1-12, Bacillus species V3-13, Bacillus timonensis, Bacillus timonensis, Bacillus weihaiensis, Bacillus yapensis, Calidifontibacillus erzurumensis, Calidifontibacillus oryziterrae, Cytobacillus luteolus, Fredinandcohnia aciditolerans, Fredinandcohnia humi, Fredinandcohnia onubensis, Fredinandcohnia onubensis, Geobacillus genomosp. 3, Geobacillus sp. 46C-IIa, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillusstearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus stearothermophilus, Geobacillus thermodenitrificans NG80-2, Halobacillus dabanensis, Halobacillus halophilus, Halobacillus halophilus, Jeotgalibacillus proteolyticus, Litchfieldia alkalitelluris, Litchfieldia salsa, Mesobacillus harenae, Metabacillus, Metabacillus litoralis, Metabacillus sediminilitoris, Oceanobacillus limi, Oceanobacillus species Castelsardo, Ornithinibacillus, Ornithinibacillus bavariensis, Ornithinibacillus contaminans, Ornithinibacillus halophilus, Ornithinibacillus scapharcae, Parageobacillus caldoxylosilyticus, Parageobacillus species, Parageobacillus thermantarcticus, Parageobacillus thermantarcticus, Parageobacillus thermoglucosidasius, Parageobacillus thermoglucosidasius, Paucisalibacillus globulus, Paucisalibacillus species EB02, Priestia abyssalis, Priestia endophytica, Priestia filamentosa, Priestia koreensis, Priestia megaterium, Psychrobacillus glaciei, SalinibacillusIt can be isolated from bacteria such as, for example, xinjiangensis, Sutcliffiella cohnii, and Thermolongibacillus altinsuensis.

[0087] The Trp RNA binding attenuator protein is a bacterial circular homo 11-mer (see A. A. Antson, J. Otridge, A. M. Brzozowski, E. J. Dodson, G. G. Dodson, K. S. Silson, T. Smith, M. Yang, T. Kurecki, P. Gollnick, hereby incorporated by reference). The structure of the trp RNA binding attenuator protein can be found in the literature (Nature 374, 693-700 (1995), hereby incorporated by reference).

[0088] Suitably, the protein used herein is a modified version of the wild-type TRAP isolated from Bacillus stearothermophilus, which can be seen in Table 1:

[0089] [Table 1]

[0090] The wild-type TRAP Bacillus stearothermophilus gene sequence can be seen in Table 2:

[0091] [Table 2]

[0092] Preferably, the preparation of the protein is carried out by biomolecule expression in a suitable expression system and purification of the expression product, preferably using a modified version of the wild-type TRAP Bacillus stearothermophilus gene sequence described above.

[0093] TRAP proteins form rings, herein "TRAP rings," which are the native state of TRAP proteins. Typically, as in the case of the Geobacillus Stearothermophilus protein demonstrated herein, TRAP monomeric proteins spontaneously assemble into circular bodies or rings made from monomers.

[0094] References herein to the "TRAP cage lumen" are the hollow interior of the TRAP cage, which is separated from the external environment by the TRAP rings that form the wall of the TRAP cage, and any holes in this wall are considered to separate the lumen from the external environment by a flat plate between the ends of the TRAP rings that lines the hole.

[0095] TRAP cages are only formed under certain conditions, for example as demonstrated herein, with the presence of cysteines that can be bridged, resulting in a ring that associates with the cage. For example, as demonstrated herein, they will form with the presence of a cysteine ​​at position 35 (as a result of the K35C mutation).

[0096] References herein to a "TRAP ring" are synonymous with a TRAP building block, a subunit of a TRAP cage complex, or a TRAP monomer assembly. References herein to an "analog" of a particular protein or nucleotide sequence refer to a protein or nucleotide sequence that has sufficient identity or homology to the protein or nucleotide sequence to be capable of performing a specified function, e.g., TRAP cage formation under conditions described herein, or to encode a protein that is capable of performing a specified function, e.g., TRAP cage formation under conditions described herein.

[0097] To determine the identity / homology percentage of two sequences, the sequence in question and the reference are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). A sequence can be determined to be an analog of a particular one if it has preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acids or nucleotides of the reference sequence of the relevant length. When comparing amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions, if a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap.

[0098] Suitably, the TRAP protein comprises an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% identity or homology to the amino acid sequence of SEQ ID NO: 1. Preferably, the TRAP protein comprises an amino acid sequence having at least 85% identity or homology to the amino acid sequence of SEQ ID NO: 1.

[0099] Reference herein to a "TRAP cage" refers to an associated protein complex formed from a plurality of biomolecules, herein a plurality of TRAP protein rings forming a complex. The TRAP protein rings may be linked together by a crosslinker, herein a molecular crosslinker. "Complex", "association", "aggregate" are used alternatively in this description to refer to a superstructure built by a reaction between biomolecules. The amount of units involved in the complex depends on the nature of the biomolecule. More specifically, it is present in the amount of biomolecule and the amount of -SH groups present in the biomolecule.

[0100] The TRAP protein is a suitable biomolecule model for the methods of the invention, likely due to its high inherent stability, cyclic shape, lack of native cysteine ​​residues (for easier control of the conjugation process), and the availability of residues that can be converted to cysteine, where the resulting cysteine ​​is in the right chemical and spatial environment for proper bond formation.

[0101] References herein to "programmable" are intended to convey that the TRAP cages of the present invention have properties imparted to them or engineered into them that make them prone or predisposed to behave in a specific and selected manner upon exposure to specific environmental conditions or stimuli.

[0102] References herein to "open" are synonymous with a TRAP cage that has fractured, leaked, fragmented, broken, or generally permitted cargo to escape from the interior of the cage.

[0103] References herein to "closed" are synonymous with a TRAP cage remaining intact, unbroken, impermeable, or generally remaining as a cage in its entirety.

[0104] References herein to "bifunctional" refer to molecular crosslinkers having two functional groups, e.g., herein, molecules having two functional groups, one functional group for each of the cysteine ​​thiol groups to be crosslinked to connect the TRAP rings in the TRAP cage. References herein to "homo-bifunctional" refer to bifunctional linkers in which the two groups are the same. Preferably, homo-bifunctional linkers include bismaleimidohexane (BMH), dithiobismaleimidoethane (DTME), bis-halomethylbenzenes and their derivatives, 2-bis-bromomethyl-3-nitrobenzene (BBN), bis-bromoxylene, and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0105] A "molecular crosslinker" is a molecule that acts to connect a unit, subunit, molecule, biomolecule, or monomer to other instances thereof through the formation of one or more chemical bonds. A molecular crosslinker is not a single atom linker that is a separate entity.

[0106] References herein to "encapsulation" within a TRAP cage are synonymous with surrounded, encased, contained, or confined by the TRAP cage.

[0107] References herein to "guest cargo" refer to the biologic, or whatever is encapsulated within the TRAP cage.

[0108] The guest cargo may be a protein, preferably selected from the group including enzymes (e.g., proteases, nucleases, hydrogenases, dehydrogenases, lipases, lyases, ligases, transferases, reductases, recombinases, nucleases acid-modifying enzymes, or other types of enzymes), antigens, antibodies. Alternatively, the cargo is another type of protein biological macromolecule (e.g., sterols, steroids, or fatty acids). Alternatively, the cargo may be a lipid, a peptide (e.g., peptide hormones, cell membrane disrupting peptides, T cell stimulating peptides, or another type of peptide), a nucleic acid (e.g., DNA, engineered DNA nanostructures, including those designed using DNA origami techniques, DNAzymes, RNA, mRNA, miRNA, siRNA, tRNA, single-stranded RNA, double-stranded RNA, RNAzymes), small molecule cargo such as drugs, peptide nucleic acids (PNAs), carbon-based structures (e.g., fullerenes or buckminsterfullerenes, single-walled or multi-walled carbon nanotubes). ), metals (e.g., iron, zinc, platinum, copper, sodium, cadmium, lanthanides, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof), toxins (e.g., ligand-targeted toxins, protease-activating toxins, melittin, and toxin-based suicide gene therapy drugs), or nanoparticles (e.g., metal nanoparticles such as gold, iron, silver, cobalt-cadmium-selenium, titanium dioxide, etc.) or core-shell metal nanoparticles such as CdS / ZnS, CdSe / ZnS, CdSe / CdS, and InAs / CdSe nanoparticles.

[0109] Enzymes include bromelain, botulinum toxin A, thrombin factor VIIA, protein C, TEV protease, specifically the SB, SC, SE, SF, SH, SJ, SK, SO, SP, SR, SS, ST, PA, PB, PC, and PE superfamilies, including Lon-A peptidase, Clp protease, lactoferrin, and nucleoporin 125, and serine proteases including the S48, S62, S68, S71, S72, S79, S81 families, specifically the CA, CD, CE, CF, CL, CM, CN, CO, CP, PA, PB.PC, PD, and PE superfamilies, including papain, cathepsin K, calpain, separase, adenine, sortase A, and Hedgehog proteins, and C7, C8, C21, C23, C4, C5, C6, ... , C27, C36, C42, C53, and C75 families; aspartic proteases including the AA, AC, AD, AE, and AF superfamilies, with specific examples such as BACE1, BACE2, Cathespin D, Cathepsin E, Chymosin, Napsin-Ad, Nepenthesin, Pepsin, Presenilin, Plasmepsin; threonine proteases including the PB and PE superfamilies, specifically including orhithine acyltransferases; glutamic proteases including the G1 and G2 superfamilies; metalloproteinases including metalloexopeptidases and metalloendopeptidases.

[0110] The enzyme can be an endonuclease, such as a nuclease selected from the group including deoxyribonuclease I; human endonuclease V, CRISPR associated proteins (including Cas9, Cas12, Cas13) with or without associated nucleic acids, including guide RNA; AP endonucleases; and flap endonucleases.

[0111] The protein may be another type of enzyme, such as SUMO-activating enzyme E1, DNA repair enzymes, such as DNA ligases, DNA methyltransferases, such as the m6A, m4C, and m5C classes, ten-eleven translocation methylcytosine dioxygenases, early growth response protein 1 (EGR1), oxoguanine glycosylases, caspases, such as pVHL, CRBN, Mdm2, beta-TrCP1, DCAF15, DCAF16, RNF114, c-IAP1, or E1 ligases, E2 ligases, DNA glycosylases, or toxins, such as ricin toxin A chain, diphtheria toxin and fragments thereof, pore-forming toxins, such as exotoxin A, α-hemolysin , Gyr-I, myeloid cell leukemia 1 (Mcl-1), DNA polymerase including DNA polymerase beta, polymerase delta, and polymerase epsilon, or enzyme replacement therapy enzymes, such as agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha.

[0112] The cargo may be anything that can act to be recognized as an antigen, e.g., SARS-CoV-2 spike protein full length, SARS-CoV-2 spike protein, receptor binding domain, SARS-CoV-2 spike protein, peptides thereof, SARS-CoV-2 spike protein full length, SARS-CoV-2 spike protein, receptor binding domain, SARS-CoV-2 spike protein, peptides thereof, SARS-CoV-2 non-spike structural protein, SARS-CoV-2 non-spike structural protein, peptides thereof ... ov-2 genome encoded protein or portion thereof, respiratory syncytial virus spike protein full length, respiratory syncytial virus spike protein, receptor binding domain, respiratory syncytial virus spike protein, peptides thereof, respiratory syncytial virus spike protein full length, respiratory syncytial virus spike protein, receptor binding domain, respiratory syncytial virus spike protein, peptides thereof, respiratory syncytial virus non-spike structural protein, respiratory syncytial virus non-spike structural protein, peptides thereof, respiratory syncytial virus a virus genome encoded protein or a portion thereof, Lassa virus spike protein full length, Lassa virus spike protein, receptor binding domain, Lassa virus spike protein, peptides thereof, Lassa virus spike protein full length, Lassa virus spike protein, receptor binding domain, Lassa virus spike protein, peptides thereof, Lassa virus non-spike structural protein, Lassa virus non-spike structural protein, peptides thereof, a Lassa virus genome encoded protein or a portion thereof, Epstein-Barr virus spike protein full length, Epstein-Barr virus spike protein, receptor binding domain, Epstein-Barr virus spike protein, peptides thereof, Epstein-Barr virus spike protein full length, Epstein-Barr virus spike protein, receptor binding domain, Epstein-Barr virus spike protein, peptides thereof, Epstein-Barr virus spike protein full length, Epstein-Barr virus spike protein, receptor binding domain, Epstein-Barr virus spike protein, peptides thereof, Epstein-Barr virus non-spike structural protein,The protein may be an Epstein-Barr virus non-spike structural protein, a peptide thereof, an Epstein-Barr virus genome encoded protein or a portion thereof, a dengue virus structural protein N, M, or E, a dengue virus structural protein N, M, or E, a peptide thereof, a dengue virus structural protein N, M, or E, a portion thereof, a cytomegalovirus protein, a portion thereof, and derived peptides including capsid proteins, tegument proteins, polymerases, and other proteins encoded by the viral genome, an influenza virus full length HA protein, an influenza virus HA protein, a receptor binding domain, an influenza virus HA protein, a peptide thereof, an influenza virus non-HA structural protein, an influenza virus non-HA structural protein, a peptide thereof, an influenza virus genome encoded protein or a portion thereof,

[0113] Cargos may be antibodies, such as anti-p53 antibodies, anti-mutant p53 antibodies, anti-JAK mAbs, such as tofacitinib and baricitinib, interleukin inhibitors, such as tocilizumab, secukinumab, and ustekinumab, anti-CD20 mAbs, such as rituximab, ofatumumab, and ocrelizumab, anti-TNF mAbs, such as infliximab, adalimumab, and golimumab, anti-IgE mAbs, such as omalizumab, hematopoietic growth factors, such as epoetin, anti-PD1 and PDL-1 mAbs, such as Keytruda, anti-CTLA4 mAbs, such as ipilimumab, anti-IL2 antibodies, anti-Il12 antibodies, anti-Il15 antibodies, anti-TGF beta antibodies, anti-angiogenic mAbs, such as Avastin, antagonist mAbs of the A2A and A2B receptors, anti-Her2 The mAb may be, for example, trastuzumab, an antibody-dependent conjugate, an anti-EGFR mAb, an anti-VEGFR mAb, an anti-CD52 mAb, for example, alemtuzumab, an anti-BAFF mAb, for example, belimumab, an anti-CD19 mAb, for example, blinatumomab, an anti-CD30 mAb, for example, brentuximab vedotin, an anti-CD38 mAb, for example, daratumumab, an anti-VEGFR2 mAb, for example, ramucirumab, or an anti-IL6 mAb, for example, siltuximab.

[0114] Proteins may be of different types, such as target of rapamycin (TOR), GATA transcription factor Gaf1 (Gaf one), TALE (transcription activator-like effector) proteins, zinc finger proteins, tumor suppressor proteins including those involved in the control of gene expression, such as p16, signal transduction factors, such as (TGF)-β; checkpoint control proteins, such as BRCA1, proteins involved in cell adhesion, such as CADM1, DNA repair proteins, such as p53, transcription factors, such as Yamanaka factors (Oct3 / 4, Sox2, Klf4, c-Myc), cytochrome c, Bcl The therapeutic agent may be a BCL protein, including B-cell lymphoma 2 (B-cell lymphoma 2), a transcriptional regulator protein, such as NF-κB, a chemokine, an interferon, an interleukin, including interleukin-2, and artificial versions thereof, a lymphokine, and a cytokine, including tumor necrosis factor, a heat shock protein, including heat shock beta-1 protein, a growth factor, such as GDF11, ubiquitin, a DNA double strand break repair protein, such as DNA ligase III alpha, a PCSK9 inhibitor, such as evolocumab and alirocumab, a brain-derived neurotrophic factor (BDNF), or an inhibitor of IL-5, such as mepolizumab and reslizumab.

[0115] The cargo may be another type of biological macromolecule (e.g., a sterol, a steroid, or a fatty acid). The sterol may be cholesterol. The steroid may be progesterone. The fatty acid may be a saturated fatty acid, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, or an unsaturated fatty acid, such as myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid.

[0116] The cargo may be a lipid such as a phospholipid, such as phosphatidylcholine, phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylserine (PS), phosphatidylinositol (PI0), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2), and phosphatidylinositol triphosphate (PIP3), (sphingomyelin) (SPH), ceramide phosphorylethanolamine (sphingomyelin) (Cer-PE), etc.

[0117] The cargo can be a peptide, such as a peptide hormone, a cell membrane disrupting peptide, a T cell stimulating peptide, or another type of peptide. The peptide hormone can be adrenocorticotropic hormone (ACTH), amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormone, follicle stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid stimulating hormone (TSH), thyrotropin releasing hormone (TRH), vasopressin, also called arginine vasopressin (AVP) or antidiuretic hormone (ADH), or vasoactive intestinal peptide (VIP). The cell membrane disrupting peptide can be melittin. The T cell stimulating peptide can be an antigen, such as a portion of an antigenic protein described above. Another type of peptide can be microcin B-17 and derivatives, albicidin and derivatives, peptide inhibitors of myeloid cell leukemia 1 (mcl-1), pepstatin and its derivatives.

[0118] The cargo may be an antibiotic molecule, such as a macrolide antibiotic, nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide, cholesterol absorption inhibitors, such as ezetimibe, fibrates, such as gemfibrozil, bezafibrate, and ciprofibrate, HMG-CoA reductase inhibitors, nitrates, such as ranolazine, ivabradine, and glyceryl trinitrate, endothelin antagonists, such as bosentan, hydralazine, minoxidil, calcium channel blockers, Antidysrhythmics such as Amlodipine, Nifedipine, Verapamil, Diltiazem, Angiotensin antagonists such as Losartan, Valsartan, Candesartan, and Irbesartan, ACE inhibitors such as Captopril, Enalapril, Lisinopril, Digoxin, Adenosine receptor agonists, Class IV Antidysrhythmics such as Verapamil, Class III Antidysrhythmics such as Amiodarone, Class II Antidysrhythmics such as Bisoprolol, Esmolol, and Propranolol, Class I Antidysrhythmics such as Fleca inides and disopyramides, antihistamines such as promethazine, cyclizine, and cetirizine, glucocorticoids such as prednisolone, dexamethasone, and hydrocortisone, antiproliferative immunosuppressants, calcineurin inhibitors such as cyclosporine, uricosurics such as allopurinol and flebuxostat, DMARDs, COX-2 inhibitors such as celecoxib, etoricoxib, and parecoxib, NSAIDs, DOPA decarboxylase inhibitors such as carbidopa or benserazide, select selective B3-adrenergic receptor agonists, a1-receptor agonists, B1 receptor agonists such as dobutamine, a1 receptor antagonists such as prazosin, doxazosin, and tamsulosin, B2 receptor agonists such as salbutamol and terbutaline, nicotinic partial agonists such as varenicline, peripheral anticholinesterases such as neostigmine, neuromuscular blockers such as panucuronium, vecuronium, and rocuronium, bladder control drugs such as oxybutynin and tolterodine, antimetabolites,For example, folate antagonists, pyrimidine analogues, and purine analogues, alkylating agents, antifungal agents such as Griseofulvin, Caspofungin, and Terbinafine, antifungal antibiotics such as Amphotericin and Nystatin, artemisinin derivatives such as Artesunate and Artemisinin, folate inhibitors such as Proguanil, Primaquine, Blood Schizonticides, schizonticides such as chloroquine and quinine, neuraminidase inhibitors such as oseltamivir and zanamivir, DNA polymerase inhibitors such as acyclovir and ganciclovir, protease inhibitors such as darunavir and ritanovir, reverse transcriptase inhibitors such as nevirapine and efavirenz, antiepileptics such as carbamazepine, gabapentin, and pregabalin, tricyclic antidepressants such as amitriptyline, nortriptyline, and desipramine, opioids, AMPA receptor blockers such as topiramate, barbiturates, benzodiazepines such as lorazepam, midazolam, and diazepam, sodium channel inhibitors such as carbamazepine, oxcarbazepine, and phenytoin, bipolar disorder medications for the treatment of mental disorders such as lithium, dopamine reuptake inhibitors such as bupropion, monoamine oxidase inhibitors such as phenelzine, isocarboxcazid, and moclobemide, noradrenaline reuptake inhibitors such as reboxetine and maprotiline, SNRIs such as venlafaxine, duloxetine, and desvenlafaxine, SSRIs such as fluoxetine, paroxetine, citalopram, escitalopram, and sertraline, tricyclics such as imipramine and clomipramine, anti-psychotics such as amisulpride and sulpiride, partial serotonin agonists, NMDA receptor antagonists such as memantine, cholinesterase inhibitors such as donepezil, rivastigmine, and galantamine,Monoxidase inhibitors such as selegiline and rasagiline, COMT inhibitors such as entacapone and tolcapone, dopamine agonists such as pramipexole and rotigotine, phosphodiesterase type V inhibitors such as sildenafil and tadalafil, uterine stimulants such as misoprostal, ergometrine, and oxytocin, GnRH analogues and inhibitors, alpha-glucosidase inhibitors dipeptidyl peptidase inhibitors such as sitagliptin, saxagliptin, and linagliptin; proton pump inhibitors such as omeprazole, lansoprazole, and pantoprazole; inhaled glucocorticoids such as neclometasone and budesonide; inhaled muscarinic antagonists such as tiotropium and glycopyrronium The cargo may be a small molecule such as leukotriene antagonists, e.g. montelukast, beta2-receptor agonists, e.g. almetrol and formoterol, anticoagulants, e.g. dabigratran, heparin, and apixaban, STING antagonists, inflammasome inhibitors, targeted oncology drugs, protein kinase inhibitors, cell cycle inhibitors, PROTACs and other promoters of protein degradation, PARP inhibitors, e.g. niraparib, ALK inhibitors, e.g. alectinib, HDAC inhibitors, e.g. belinostat, MEK inhibitors, e.g. cobimetinib, BRAF inhibitors, e.g. dabrafenib, EGFR inhibitors, e.g. erlotinib, mTOR inhibitors, e.g. everolimus, HER2 inhibitors, e.g. lapatinib, FLT3 kinase inhibitors, e.g. midostaurin, JAK inhibitors, e.g. tofacitinib, or BCL2 inhibitors, e.g. venetoclax. ,

[0119] "Unit," "subunit," "molecule," "biomolecule," and "monomer" are used alternatively in this description to mean one molecule that connects to another molecule to form a complex.

[0120] References herein to "reduction-resistant / insensitive molecular crosslinkers" are references to crosslinkers that are not cleaved by reduction reactions such as those typically seen when disulfide bonds are cleaved by reducing agents. These crosslinkers are stable under conditions that would result in rupture of reduction-sensitive bonds. These include bismaleimidohexane (BMH) and bis-bromoxylene.

[0121] References herein to "reduction-responsive / sensitive molecular crosslinkers" are references to crosslinkers that are cleaved by a reduction reaction, such as those typically seen when disulfide bonds are cleaved by reducing agents. These crosslinkers are not stable under conditions that would result in rupture of reduction-sensitive bonds. These include dithiobismaleimidoethane (DTME).

[0122] References herein to "photoactivatable molecular crosslinkers" are to crosslinkers that are photoreactive or photosensitive, i.e., that will be cleaved when exposed to light. The light can be UV or other such light of a specific range of wavelengths. These include 2-bis-bromomethyl-3-nitrobenzene (o-BBN), 2,4-bis-bromomethyl-1-nitrobenzene (m-BBN), and 1,3-bis-bromomethyl-4,6-dinitro-benzene (BDNB).

[0123] Reference herein to the lumen of a "highly charged" TRAP cage means that the surface facing the lumen of the cage undergoes a net change in charge equivalent to at least +1 or -1 per TRAP ring, compared to the non-mutated (wild-type) ring. Thus, a 24-ring TRAP cage, if made highly charged, will have a minimum change in charge of -24 or +24, compared to a variant that is not highly charged.

[0124] Additionally, the following abbreviations are used: TRAP (trp RNA-binding attenuation protein), GFP (green fluorescent protein), PTD4 (protein transduction domain), CPP (cell-penetrating peptide), SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), TEM (transmission electron microscopy), DMEM (Dulbecco's modified Eagle's medium), and FBS (fetal bovine serum).

[0125] The transport of molecular cargo into cells is desirable for a wide range of applications, including the delivery of drugs, genetic materials, or enzymes. Several nanoparticles have been employed to achieve this, including liposomes, virus-like particles, non-viral protein cages, DNA origami cages, and inorganic nanoparticles, each with their own advantages and disadvantages. Protein cages are a promising approach, demonstrated by viruses in nature, that can deliver genetic materials into cells, often with high efficiency and specificity.

[0126] Artificial cages are constructed by proteins that do not naturally form cage structures, and the interactions between the constituent proteins can be modified to promote their association. The advantage of using such an approach is that the resulting cages can be endowed with properties and capabilities that may be unavailable or unfeasible in their naturally occurring forms. To date, several artificial protein cages have been produced, including tandem fusions of proteins with two- and three-fold rotational symmetry that can form a 12-subunit tetrahedral cage, a 24-subunit nanocube structure with octahedral symmetry, a 60-subunit icosahedral cage structure that self-assembles from trimeric protein building blocks, and a co-associating two-component 120-subunit icosahedral protein complex comparable to that of a small viral capsid, as well as designed peptides that can form a network that closes to form a cage. Artificial protein cages have been developed to bind siRNA, mRNA, and other proteins, and to allow the formation of complexes that can be used to bind siRNA, mRNA, and other proteins. 2There are several examples in which artificial cages have been loaded with various cargos, including , and fluorescent dyes. However, only a few cases have demonstrated delivery of cargo to cells by artificial cages. To the best of our knowledge, delivery of protein / therapeutic cargo to cells mediated by artificial protein cages (as opposed to natural cages) has not been demonstrated previously.

[0127] We have previously produced an artificial protein cage, termed the TRAP cage, using a building block consisting of the naturally occurring ring-shaped protein TRAP (trp RNA-binding attenuating protein) (Figure 1a). 1 In nature, TRAP is involved in the control of tryptophan synthesis and has been well characterized structurally and biochemically. It is also used as a versatile building block in bio-nanoscience. The TRAP cage consists of 24 TRAP rings forming a diameter of approximately 22 nm, a 2.2 MDa hollow sphere with a lumen of roughly 16 nm in diameter. Each TRAP ring in the cage is connected to five TRAP ring neighbors, and the structure contains six angular holes of approximately 4 nm in diameter. Unusually, compared to other natural and most artificial cages, the ring subunits in the cage are not held together by a network of protein-protein interactions. Instead, a single gold(I) ion bridges the opposing sulfurs of cysteine ​​residues between the rings in a protein in which the naturally occurring lysine at position 35 has been replaced by a cysteine. The cysteines for 10 of the 11 monomers of each ring in the cage are bridged in this manner, leaving the 11th unbridged and available for reaction, for example with a maleimide-labeled dye.

[0128] The TRAP cage is extremely stable, surviving temperatures of 95° C. for at least 3 hours and high levels of denaturants such as urea. Despite this high stability, the TRAP cage readily disintegrates in the presence of low concentrations of reducing agents, including the cellular reducing agent glutathione. This attribute raises the prospect that the TRAP cage may have utility as a system for delivering cargo to cells, since it would be expected to retain its structure and protect the cargo until entering the cell, where intracellular reducing agents would result in disassembly and subsequent cargo release.

[0129] We show that the TRAP cage can be deliberately loaded with protein cargo, using the highly negatively charged variant of green fluorescent protein, GFP(-21), as an example molecule. We also show that the TRAP cage can be used to deliver such cargo to the interior of human cells. This cell penetration is itself controllable, since it only occurs if the surface of the TRAP cage is modified, for example, by a cell-penetrating peptide. The results are a first step towards the development of the TRAP cage as a potentially useful tool for delivering medically relevant cargo to cells, and more broadly demonstrate the potential of engineered protein cage systems as therapeutic agents.

[0130] Herein, we show that the TRAP cage can be used to purposefully encapsulate protein cargo and deliver it to the interior of cells. The TRAP cage employed either in its unmodified form or externally decorated showed no significant effect on cell viability.

[0131] In the first example, cargo loading was achieved using our previously developed TRAP cage, which has a positively charged patch on its interior to electrostatically capture the highly negatively charged GFP through diffusion into the cage. 1This was achieved using . Attempts to deliver loaded cargoes into cells showed no evidence of penetration of the TRAP cage into cells if they were not decorated. In contrast, attachment of the cell-penetrating peptide (CPP) PTD4 to the exterior of the TRAP cage resulted in significant penetration into the cell interior.

[0132] A small amount of previous work on engineered protein cage-mediated delivery of cargo to cells has demonstrated success for non-protein cargo. Remarkably, it has been shown that engineered protein cages loaded with siRNA can be taken up by a variety of mammalian cells and release their cargo to induce RNAi and knockdown of target gene expression. 3 In this case, the high gene silencing efficiency combined with low toxicity effects indicated that the protein cage carrier has potential as a therapeutic delivery system. Encapsulation of protein cargos within engineered protein cages has been demonstrated previously. However, these cages were not shown to be able to deliver their cargos directly to cells; instead, multi-copy cages themselves were used as cargos within lipid envelopes created in cells and purified as encapsulated protein nanocages (EPNs), where the lipid envelope was derived from the host cell membrane. This meant that the EPNs could deliver the cages and entry into the cells was achieved by the encapsulating host-derived membrane, not the protein cage.

[0133] The overall high stability of the TRAP cage, but its demonstrated ability to disassemble in the presence of cellular reducing agents 1Given this, it would be interesting to know whether the cage would easily break apart once inside the cell. The change in the relative signal intensity of Alexa-647 versus GFP bound to the TRAP cage upon entry into the cell suggests the intracellular disintegration of the cage and release of the cargo. A possible explanation is that when Alexa647 and GFP are in close proximity to each other due to binding to the TRAP cage, GFP fluorescence may decrease due to a quenching effect from the dye. Once GFP is released by TRAP cage disassembly, the average distance from GFP to Alexa-647 becomes larger, resulting in an increase in the detected GFP fluorescence. This possibility is supported by the observation that the signal from intracellular GFP is visibly brighter than when it is delivered using a TRAP cage lacking Alexa-647 (Figure 10).

[0134] Overall, the work presented here provides the first demonstration of protein delivery to cells mediated by an engineered protein cage. The demonstrated cargo loading efficiency is rather low, which may be addressed by further modifying the TRAP cage to carry a higher density of positive charges inside the cage.

[0135] Furthermore, loading of functional protein cargo into the TRAP cage is demonstrated using genetic fusion of the cargo to the lumen-facing N-terminus of the TRAP monomer. The patchwork expression approach allows for mixed expression of TRAP monomers without fusions or allowing control of the amount of cargo so that it does not exceed the capacity of the cage. In this way, TRAP cages with mCherry cargo as well as with a mixture of mCherry and mOrange cargo are demonstrated and the presence of the cargo is confirmed.

[0136] Furthermore, the genetic fusion approach was adapted to provide multiple copies of the lumen-facing SpyCatcher protein, which was demonstrated to be capable of capturing the functional proteins green fluorescent protein (GFP) and a computationally designed interleukin-2 mimetic (neoleukin-2 / 15, NL-2) bearing a SpyTag (Silva DA et al., Nature, 2019, 565, 186-191). We further showed that the interaction of NL-2 with its target cell receptor was significantly inhibited by encapsulation within the TRAP cage, and that when the TRAP cage was opened by an exogenously applied trigger, the cargo became functional at a level similar to free NL-2.

[0137] Alternatively, various methods of cargo capture (such as covalent attachment) can be explored, as described for other protein cages. 4,5Additionally, we expect further modifications of the TRAP cage to both increase targeting specificity and broaden the scope and usefulness of encapsulated cargo. Finally, future investigations will be required to pinpoint and track both the precise intracellular location of the TRAP cages and their quaternary state. According to an embodiment, the cages described herein can be used as medicines. This can be in treating a patient, such as administering the cages described herein to a patient, or including the cages described herein for use in treating a disease in a patient. This can be specifically a cage designed to carry cargo and disassemble in the presence of a reducing agent for intracellular delivery. These cages can be administered with or in the presence of a pharma- ceutical acceptable carrier, adjuvant, or excipient. Cages for use as medicines or for treating a patient would be beneficial to said patient. For example, as a drug delivery system (DDS) for active molecules (biological macromolecules such as RNA, DNA, peptides, and proteins, among others), they offer advantages because biological macromolecules are often easily destroyed or digested by conditions such as those found in vivo. Biological macromolecules are too large to diffuse through the holes in TRAP, which is a large protein, and the TRAP cage can undergo significant changes without destroying the overall structure. This means that it can be modified to capture a therapeutic cargo and simultaneously modified externally to target a therapeutic target. A programmable linker can be used that cleaves in a desired situation that correlates with arrival at the site of action. For example, light can be shone on the target site to cleave the open photocleavable TRAP cage. When the TRAP cage penetrates a cell, those tethered by the reductive linker will spontaneously open and release the cargo as the cell's cytoplasm becomes highly reducing. The cage may also be used in conjunction with or to act as a vaccine, where an antigen (i.e., a peptide) expected to stimulate a T cell response is captured inside the TRAP cage and then targeted to the T cell, followed by triggered opening. [Brief description of the drawings]

[0138] [Figure 1] Diagram of the TRAP cage protein. (a) Structure of the TRAP cage (PDB: 6RVV) with each TRAP ring shown in a different color. Gold atoms are shown as yellow spheres. (b) Surface representation of the TRAP cage exterior (left) and interior (right) colored by charge distribution. (c) Surface image of a single TRAP ring with the face pointing towards the interior cavity shown, colored according to charge. (d) Highly negatively charged GFP(-21) shown in cartoon representation (left) and surface image colored according to charge (right). (e) Scheme of TRAP cage encapsulation with GFP(-21) and external modification with Alexa-647 dye and PTD4 peptide. [Diagram 2]Diagram of TRAP cage loading and decoration. (a) Native PAGE gel showing purified TRAP cages incubated with His-tagged GFP(-21) after passing through a Ni-NTA column in the absence (-TCEP) or presence (+TCEP) of TCEP. Lane 1: GFP(-21) positive control; 2: molecular weight marker for native PAGE; 3: empty TRAP cage; 4: input (TRAP cage with GFP(-21)); 5 and 8: flow-through; 6 and 9: wash; 7 and 10: elution. Collected fractions were stained for protein (left) or analyzed by fluorescence detection (right, excitation 488 nm). (b) Collected fractions were subjected to SDS-PAGE followed by Western blot with anti-GFP detection. Lane 1: GFP(-21) positive control; 2: molecular weight markers for SDS-PAGE; 3: empty TRAP cage; 4: input (TRAP cage with GFP); 5 and 8: flow-through; 6 and 9: wash; 7 and 10: elution. (c) Native PAGE gel showing encapsulation of GFP(-21) by unmodified TRAP cage or TRAP cage externally modified with Alexa-647 and PTD4. Lane 1: TRAP cage with GFP(-21); 2: TRAP cage with GFP(-21) decorated with Alexa-647; 3: TRAP cage with GFP(-21) decorated with Alexa-647 and PTD4; 4: molecular weight markers for native PAGE. Gels were stained for protein (top panel) and analyzed by fluorescence detection of GFP (middle panel, excitation 488 nm) and Alexa-647 (bottom panel, excitation 647). (d) Negative staining transmission electron microscopy of a TRAP cage with GFP(-21) (left panel); a TRAP cage with GFP(-21) decorated with Alexa-647 (middle panel); a TRAP cage with GFP(-21) decorated with Alexa-647 and PTD4 (right panel). [Diagram 3]Diagram of delivery of TRAP cage bearing GFP(-21) into MCF-7 cells. (a) Representative flow cytometry dot plots for MCF-7 cells after 4 h treatment with Alexa-647-labeled TRAP cage bearing GFP(-21) (represented as (TC+GFP)+Alexa-647) and after 15 min, 2 h, and 4 h treatment with TRAP cage bearing GFP(-21) labeled with Alexa-647 and PTD4 peptide (represented as (TC+GFP)+Alexa-647+PTD4). The x-axis and y-axis indicate the fluorescence intensity of GFP and Alexa-647, respectively. Untreated cells were used as a negative control. (b) Representative red and green fluorescence overlay histograms for MCF-7 cells from the same experiment. (c) Median fluorescence intensity of Alexa-647 and GFP positive cells treated with TRAP cages bearing GFP and decorated with Alexa-647 or decorated with both Alexa-647 and PTD4 after 15 min, 2 h, and 4 h of incubation. Data are normalized to untreated cells and are based on three independent experiments. Controls: 1: untreated cells; 2: cells incubated with (TC+GFP)+Alexa-647. (d) Confocal microscopy images of untreated cells (control cells, top row), cells incubated with TRAP cages loaded with GFP(-21) and labeled with Alexa-647 only (middle row); and cells incubated with TRAP cages loaded with GFP(-21) and labeled with Alexa-647 and PTD4 (bottom row). Actin filaments were stained with phalloidin conjugated to Alexa-568, and nuclei were stained with DAPI. Green channel-GFP; red channel-Alexa-647; blue channel-DAPI; grey channel-Alexa-568; (Scale bar: 10 µM). [Figure 4]Tracking of TRAP cage and GFP(-21) in MCF-7 cells. Confocal microscopy merged images of cells incubated with TRAP cage carrying GFP(-21) decorated with Alexa-647 and PTD4 and fixed at various time points. Actin was stained with phalloidin conjugated to Alexa-568, while DAPI was used for nuclear staining; (Scale bar: 10 μM). Rectangular images below each main image are representative orthogonal images in yz axis. (a) - Image with red channel maximum projection; (b) - Image with green channel maximum projection. [Diagram 5] Estimation of the number of His-tagged GFP(-21) molecules in the TRAP cage. (a) Standard curve obtained from fluorescence measurements of GFP(-21) protein with a concentration range of 0-100 nM. Fitted with the equation: y=0.0258x+4.4; R2=0.9786. (b) Western blot used for band densitometry analysis. Lanes 1-4: GFP(21); lane 5: TRAP cage carrying GFP(-21) (denoted as (TC+GFP)). [Figure 6]Diagram of external decoration of TRAP cage with GFP(-21). (a) RP-HPLC chromatogram showing purified PTD4 peptide used to decorate TRAP cage loaded with GFP(-21). (b) Native PAGE gel showing TRAP cage carrying GFP(-21) after titration of Alexa-647 in the conjugation reaction. Gels were analyzed by fluorescence detection of Alexa647 (left panel, excitation 647) and GFP (middle panel, excitation 488 nm) and stained for protein (right panel). Arrows indicate optimal decoration conditions used in further experiments. (c) SDS-PAGE gel comparing TRAP cage carrying GFP(-21) without decoration, decorated with Alexa-647, or decorated with both Alexa-647 and PTD4. Left: detection at 488 nm; middle: detection at 647 nm; right: Western blot for the same samples detected with anti-GFP antibody. Lanes: 1: molecular weight marker for SDS-PAGE electrophoresis; 2: TRAP cage with GFP(-21); 3: TRAP cage with GFP(-21) decorated with Alexa-647; 4: TRAP cage with GFP(-21) decorated with Alexa-647 and PTD4; 5: GFP(-21)-positive control. [Figure 7] Figure 1. TRAP cage stability in culture medium and cell viability test. (a) Native PAGE gel showing TRAP cage stability in DMEM culture medium with or without FBS presence during 18 hours of incubation. (b) Cell viability of MCF-7 and HeLa cells after 4 hours of exposure to empty TRAP cage, TRAP cage loaded with GFP(-21), and TRAP cage with GFP(-21) decorated with Alexa-647 and PTD4. M=molecular weight marker for native electrophoresis; TC: empty TRAP cage; (TC+GFP): TRAP cage loaded with GFP(-21); (TC+GFP)+Alexa-647+PTD4: TRAP cage with GFP(-21) and decorated with Alexa-647 and PTD4. [Figure 8]Diagram of delivery of TRAP cage with GFP(-21) into HeLa cells. (a) Representative flow cytometry dot plots for HeLa cells after treatment with Alexa-647-labeled TRAP cage with GFP(-21) (denoted as (TC+GFP)+Alexa-647) for 4 h, and after treatment with Alexa-647-labeled TRAP cage with GFP(-21) and PTD4 (denoted as (TC+GFP)+Alexa-647+PTD4) for 15 min, 2 h, and 4 h. The x- and y-axes indicate the fluorescence intensity of GFP and Alexa-647, respectively. Untreated cells were used as negative control. (b) Representative red and green fluorescence overlay histograms for HeLa cells from the same experiment. (c) Median fluorescence intensity of Alexa-647 and GFP positive cells treated with (TC+GFP)+Alexa-647 and (TC+GFP)+Alexa-647+PTD4 after 15 min, 2 h, and 4 h of incubation. Data are normalized to untreated cells and are based on three independent experiments. Controls: 1: untreated cells; 2: cells incubated with (TC+GFP)+Alexa-647. (d) Confocal microscopy images of untreated cells (control cells) (top row), cells incubated with (TC+GFP) labeled with Alexa-647 only (middle row): cells incubated with TRAP cages loaded with GFP(-21) and labeled with Alexa-647 and PTD4 (bottom row). Actin filaments were stained with phalloidin conjugated to Alexa-568 and nuclei were stained with DAPI. Green channel-GFP; Red channel-Alexa-647; Blue channel-DAPI; Grey channel-Alexa-568; (Scale bar: 10 μM). [Figure 9]Tracking of TRAP cage and GFP in HeLa cells. Confocal microscopy merged images of cells incubated with TRAP cage carrying GFP(-21), labeled with Alexa-647 and PTD4 and fixed at various time points. Actin was stained with phalloidin conjugated to Alexa-568, while DAPI was used for nuclear staining; (Scale bar: 10 μM). Rectangular images are representative orthogonal images in yz axis. (a) - Image with red channel maximum projection; (b) - Image with green channel maximum projection. [Figure 10] Diagram of the effect of Alexa-647 on GFP(-21) fluorescence. (a) Cells were exposed to Alexa-647 and PTD4 labeled (TC+GFP) (top row) or to PTD4 only (TC+GFP) (bottom row). Actin filaments were stained with phalloidin conjugated to Alexa-568 and nuclei were stained with DAPI. Green channel - GFP; Red channel - Alexa-647; Blue channel - DAPI; Grey channel - Alexa-568; (Scale bar: 10 μM). (b) Mean GFP fluorescence intensity registered from three different fields of view for samples exposed to cells labeled with Alexa-647 and PTD4 (TC+GFP) or to PTD4 only (TC+GFP). Fluorescence intensity was quantified using ImageJ, taking into account background intensity subtraction. (c) Mean fluorescence of GFP(21) encapsulated in undecorated and fully decorated TRAP cages measured in solution. [Figure 11] Diagram of guest packaging of a single type of guest protein using genetic fusion and patchwork formation. (a) Schematic diagram of mCherry encapsulation into TRAP cages using genetic fusion and patchwork strategies. Ptet / tetO, tetracycline promoter / operon; Pt7 / lacO, T7 promoter / lac operon system. (b) Negative staining transmission electron microscopy images of TRAP cages containing varying numbers of mCherry in the lumen. [Figure 12]Diagram of guest packaging of two different types of guest proteins using genetic fusion and patchwork formation. (a) Schematic of TRAP cages loaded with fluorescent proteins. Patchwork TRAP rings fused at the N-terminus with either mCherry (dark cylinder) or mOrange2 (light cylinder) were mixed with either DTME or triphenylphosphine monosulfate (TPPMS)-Au(I)-Cl. (b) Native PAGE showing the fluorescence properties of purified TRAP cages coupled with fluorescent cargo. Gels were visualized using InstantBlue protein staining (left) and fluorescence with excitation at 532 nm and emission at 610 nm (right). (c) TEM images of empty (left) TRAP cages and those loaded with fluorescent proteins (right) assembled using either Au(I) (top) or DTME (bottom). Scale bar, 50 nm. [Figure 13] Figure 1. Confirmation of dual protein loading in TRAP cages. a, b, Normalized emission spectra of TRAP cage Au(I) (a) and TRAP cage DTME (b) loaded with both mOrange2 and mCherry, upon excitation at 510 nm, before and after addition of 10 mM DTT. mOrange2 emission peak at 568 nm, mCherry emission peak at 610 nm. Additional lines show the spectra of cages loaded with only mOrange2 or mCherry proteins, mixed together immediately before measurement, in the absence or presence of DTT, respectively. [Figure 14] Diagram of the guest packaging concept using the SpyTag / SpyCatcher system. (a) Strategy for SpyTag / SpyCatcher-mediated guest loading. (b) Constructs of TRAP and GFP variants. SpyT, SpyTag; SpyC, SpyCatcher; contains SpyCatcher between residues 47 and 48. The construct was produced with His-tagged SUMO and cleaved by SUMO protease after Ni-NTA affinity chromatography to yield TRAP-K35C-loop SpyC. [Figure 15] Diagram of production of TRAP cages containing SpyCatcher in the lumen. (a) SDS-PAGE analysis of the production of TRAP 11mers composed of TRAP-K35C and either SpyC-TRAP or TRAP-looped SpyC. SN, supernatant after cell lysis and centrifugation; Ni, Ni-NTA affinity chromatography; SU, SUMO protease cleavage. (b) Native PAGE analysis of cage formation with Au(I). Reactions were carried out in 50 mM sodium phosphate buffer (pH 8.0) containing 100 μM TRAP, 0 or 100 μM TPPMS-Au(I)-Cl (Au(I)(-) or (+)), and 0 or 600 mM NaCl (NaCl(-) or (+)). [Figure 16] Diagram of GFP encapsulation in TRAP cages using the SpyTag-SpyCatcher system. (a, b) SDS-(a) and native-(b) PAGE analysis of mixtures of TRAP cages containing a SpyCatcher moiety in the lumen, SpyC-TRAP cages or TRAP-Loop SpyC cages, and SpyTag-GFP. For native PAGE, the same gels were visualized by fluorescence and Instant Blue staining. [Figure 17] Isolation and imaging of GFP-loaded TRAP cages. (a, b) Size-exclusion chromatogram (a) and negative stain TEM image (b) for TRAP cages containing a SpyCatcher moiety in the lumen acquired with 30 ng / mL tetracycline induction and mixed with SpyTagged GFP. A cage composed of the TRAP-K35C variant, with no cargo loaded, is also provided for comparison. (c) Negative stain TEM image of the SpyT-TRAP variant. [Figure 18]Diagram of the strategy for encapsulation of Neoleukin-2 / 15 in light-gated TRAP cages. The patchwork TRAP ring is composed of a TRAP variant containing K35C, R64S mutations and lacking lysines at positions 73 and 74, and one containing a K35C mutation, a His tag and SUMO at the N-terminus, and SpyCatcher in the luminal loop. BBN, 1,2-bisbromomethyl-3-nitrobenzene; β-ME, β-mercaptoethanol. [Figure 19] Figure 2. Induced release of neoleukin-2 from TRAP cages stimulates target cells. a, Graph reflects SEAP activity as indicated by absorbance measured after 24 h stimulation of HEK-Blue cells with NL-2, hIL-2, and Spy-Tag-NL-2 conjugated to SpyCatcher-TRAP rings; b, SEAP activity after 24 h stimulation with NL-2, empty TRAP cages, and SpyCatcher-TRAP cages loaded with SpyTag-NL-2, before and after UV irradiation. EXAMPLES

[0139] Techniques Employed in Realizing the Invention Electron microscopy GFP(-21)-loaded TRAP cages, GFP(-21)-loaded and Alexa-647-labeled TRAP cages, and GFP(-21)-loaded and fully decorated TRAP cages were imaged using a transmission electron microscope. Samples were typically diluted to a final protein concentration of 0.025 mg / ml, centrifuged at 10000 g for 5 min at room temperature, and the supernatant was applied to hydrophilized carbon-coated copper grids (STEM Co.). Samples were then negatively stained with 3% phosphotungstic acid, pH 8, and visualized using a JEOL JEM-2100 instrument operated at 80 kV.

[0140] Flow cytometry For TRAP cage internalization experiments, MCF-7 and HeLa cells were cultured at 2.5 x 10 per well. 5 Cells were seeded at a density of 10x in 12-well plates (VWR) in 800 μl DMEM with 10% FBS and cultured for an additional 16 h before the experiment. Cells were then incubated with 50 μg (6 nM) of cargo-loaded TRAP cages labeled with Alexa-647 alone or decorated with Alexa-647 and PTD4 peptides in 50 mM HEPES with 150 mM NaCl pH 7.5 supplemented with 10% FBS for 15 min, 2 h, and 4 h. After incubation, cells were washed three times for 5 min with phosphate-buffered saline (PBS) (EURx) and harvested using trypsin (1 mg / ml) and centrifuged at 150 g for 5 min. Cells were then washed three times in PBS by centrifugation (150 g for 3 min) and resuspended in PBS. Cells were run on a Navios flow cytometer (Beckman Coulter) and fluorescence of 12,000 cells was collected for each sample. Untreated cells and cells treated with cargo-loaded TRAP cages labeled with Alexa-647 only were used as negative controls. Data acquired for three independent experiments were analyzed using Kaluza software (Beckman Coulter). The percentage of Alexa-647 / GFP positive cells and median fluorescence intensity were determined for each sample.

[0141] Laser scanning confocal microscopy For fluorescence laser scanning confocal microscopy, cells were grown on 15-mm glass cover slips plated in 12-well plates (2.5 × 10 cells per well in 800 μl DMEM with 10% FBS). 5Cells were then washed with PBS (3 times for 5 min), fixed with 4% paraformaldehyde solution (15 min, room temperature), and permeabilized with 0.5% Triton-X100 in PBS (7 min, room temperature). Actin filaments were stained with phalloidin conjugated to Alexa-568 in PBS (1:300, Thermo Fisher Scientific, 1.5 h, room temperature). Coverslips were then mounted on slides using Prolong Diamond medium with DAPI (Thermo Fisher Scientific). Fluorescent images were acquired under an Axio Observer.Z1 inverted microscope (Carl Zeiss, Jena, Germany) equipped with an LSM 880 confocal module with a 63× oil immersion objective. Images were processed using ImageJ 1.47v (National Institutes of Health, USA).

[0142] Example 1. Loading of TRAP cages To pack the TRAP cage, we exploited the fact that the only significant patches of positive charge on the surface of the TRAP ring are on the faces lining the interior of the cage (Figure 1a-c, 1b, c). In principle, this could be done with a simple 1D filling technique, as has been demonstrated for other protein cages (e.g., 6 ), which may allow for the capture of negatively charged cargo via electrostatic interactions. The fact that the constituent TRAP rings do not associate into the TRAP cage until the addition of gold(I) 1This means that protein cargoes smaller than approximately 4 nm have two possible routes to encapsulation: they can bind to the TRAP ring before association, or they can be added after TRAP cage formation and allowed to diffuse into the cage through the four-fold hole. We chose the highly negatively charged GFP(-21) as a model cargo (Fig. 1d). This cylindrical protein has a diameter of approximately 2.4 nm and is therefore expected to be able to diffuse into the associated TRAP cage (Fig. 1e). His-tagged GFP(-21) was mixed with the TRAP cage and incubated overnight, followed by size-exclusion chromatography purification for removal of remaining free GFP(-21). The two proteins were found to associate, as shown by co-migration of the fluorescent signals on the native gel (Fig. 2a). To verify whether His-tagged GFP(-21) was inside the TRAP cage and not bound to its exterior, we performed a pull-down assay using Ni-NTA affinity chromatography followed by Western blot analysis. The observation that TRAP cage-bound GFP(-21) did not bind to the Ni-NTA column suggested successful encapsulation making the His-tag inaccessible. This was further supported by the pull-down assay, which showed that only bound GFP(21) was available to interact with the Ni-NTA column after the cage was dissociated by the addition of a reducing agent (Figure 2b). These results strongly suggest encapsulation of GFP in the TRAP cage in either a complete or partial manner (partial encapsulation is when GFP “blocks” the hole in the TRAP cage with the His-tag facing inward). The number of GFP(-21) per cage was approximately 0.3, comparable to that found in several other packed protein cages, although some displayed significantly higher numbers of cargo.

[0143] Production and purification of TRAP cages loaded with GFP(-21) TRAP cage production and purification was carried out as previously described.1 For relevant plasmid and amino acid sequence information, see Table 1. The highly charged (21)His tagged GFP protein was expressed from pET28a encoding the GFP gene and produced in BL21(DE3) cells. The protein was purified using Ni-NTA. Briefly, cells were lysed by sonication at 4°C in 50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2 in the presence of protease inhibitors (Thermo Fisher Scientific), and the lysate was centrifuged at 20000g for 0.5 h at 4°C. The supernatant was purified using Ni-NTA pre-equilibrated in 50 mM Tris, pH 7.9, 150 mM NaCl, 20 mM imidazole (buffer A). 2+ The protein was incubated with agarose beads coupled with bound nitrilotriacetic acid (His-Pur Ni-NTA, Thermo Fisher Scientific). After three washes of the resin (with buffer A), the protein was eluted with 50 mM Tris, pH 7.9, 150 mM NaCl, 300 mM imidazole (buffer B). Fractions containing His-tagged GFP(-21) were pooled and subjected to size-exclusion chromatography on a HiLoad 26 / 600 Superdex 200 pg column (GE Healthcare) in 50 mM Tris-HCl, pH 7.9, 150 mM NaCl at room temperature. Protein concentration was measured using a Nanodrop spectrophotometer using a wavelength of 280 nm.

[0144] GFP encapsulation was performed by mixing equal volumes of 100 μM highly negatively charged (-21)His-tagged GFP with 1 μM preformed TRAP cages incubated overnight in 50 mM Tris, 150 mM NaCl, pH 7.9. Purification of GFP-loaded TRAP was performed by size-exclusion chromatography using a Superose 6 Increase 10 / 300 column (GE Healthcare) in 50 mM HEPES, pH 7.5, 150 mM NaCl. Fractions containing TRAP cages were collected and analyzed by native PAGE using 3-12% native Bis-Tris gels (Life Technologies) followed by fluorescence detection using a Chemidoc detector (BioRad) with excitation at 488 nm.

[0145] Estimating the number of His-tagged GFP(-21) molecules in the TRAP cage To estimate GFP(-21) loading, two methods were used: 1. Based on the detection of GFP fluorescence in cargo-loaded TRAP cages. A GFP (21) standard curve was constructed with concentrations ranging from 0 to 100 nM. λ em 1.0 nm interval, scanning speed 6000 nm, λ ex Bandwidth 5nm and λ em Fluorescence spectra were acquired at 26°C using a Shimadzu RF-6000 spectrofluorometer with a fixed excitation wavelength at 488 nm and an emission wavelength range of 495-550 nm using a bandwidth of 5 nm. em Fluorescence at 510 nm was used for calculation. TRAP protein concentration was determined from absorbance at 280 nm. A TRAP cage:GFP(-21) stoichiometry of 1:0.28±0.07 was obtained (FIG. 5a).

[0146] 2. Densitometric analysis. Briefly, a series of His-tagged GFP(-21) dilutions (0.4ng; 0.8ng; 4ng; 8ng as measured by Nanodrop at 280nm) and sample cargo-loaded TRAP cage (2μg as measured by Nanodrop at 280nm) were separated by SDS-PAGE and subjected to Western blotting (Figure 5b). The signal from His-tagged GFP(-21) protein was detected using anti-GFP antibody and secondary HRP-conjugated antibody in a chemiluminescence detector (Chemidoc, BioRad). Densitometric analysis of the resulting blot using ImageLab (BioRad) software showed that 0.6ng of His-tagged GFP(-21) was present in 2μg cargo-loaded TRAP cage. Densitometric analysis resulted in a TRAP cage:GFP(-21) stoichiometry of approximately 1:0.4.

[0147] Ni-NTA "Pull Down" Samples of purified TRAP cages loaded with His-tagged GFP(-21) protein were split into two portions and incubated under reducing (1 mM TCEP) or non-reducing (no TCEP) conditions. Samples were then passed through Ni-NTA resin (Thermo Fisher Scientific) under gravity flow, and 100 μl of each sample was introduced into 50 μl of resin equilibrated with buffer A. Three samples were collected: (i) flow-through, (ii) wash with buffer A, and (iii) elution with buffer B. Samples were analyzed by native PAGE followed by fluorescence detection (excitation at 488 nm, Chemidoc, BioRad) and Western blot. For SDS-PAGE and Western blot, samples collected from Ni-NTA pull-down assays were denatured by addition of TCEP (final concentration 0.1 mM) and boiling for 15 min, followed by separation by Tris / glycine gel electrophoresis. The gel was subjected to electrophoretic transfer (2 h, 90 V) to an activated PVDF membrane in 25 mM Tris, 192 mM glycine, 20% methanol buffer. The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS-T) supplemented with 0.05% Tween 20, followed by incubation with mouse monoclonal anti-GFP antibody (1:2500; St. John's Laboratories, UK) and anti-mouse conjugated with horseradish peroxidase (1:5000, Thermo Fisher Scientific) secondary antibodies for 1.5 h. Signals were developed using Pierce ECL Blotting Substrate (Thermo Fisher Scientific) and visualized in a BioRad Chemidoc detector.

[0148] [Table 3]

[0149] The TRAP cage herein may have a highly charged lumen. To this end, the TRAP cage may include E48Q or E48K mutations. Preferably, the TRAP cage with a highly charged lumen will include K35C / E48Q or K35C / E48K mutations. This provides an additional.

[0150] Example 2. Decoration of the TRAP cage with fluorescent dyes and with cell-permeable peptide labels We aimed to modify the TRAP cage to facilitate its cell entry. We selected an optimized TAT-based cell-penetrating peptide, PTD4 (YARAAARQARA, SEQ ID NO: 8), which is more amphipathic with a reduced number of arginines and increased α-helical content, and shows significantly improved ability to penetrate cell membranes. 7 Some work has shown that coating nanoparticles with PTD4 or similar enhances cell penetration (e.g., 8 ). We attached the PTD4 derivative Ac-YARAAARQARAG (SEQ ID NO: 9) to the amino groups on the surface-exposed lysines of the TRAP cage. There are three such surface-exposed lysines per monomer on the TRAP cage, potentially allowing 792 peptides to be attached per cage. Acetylation of the N-terminal amino groups eliminates the possibility of cross-reaction of those amino groups with activated carboxyl moieties that are intended to react with available amino groups of the TRAP protein. Additionally, the extended C-terminal glycine residue serves as a flexible linker and, since it is not a chiral amino acid, discards the chance of racemization during carboxyl activation. Peptides were synthesized using solid-phase methodology and purified by reversed-phase high-performance liquid chromatography (Figure 6a). In the optimized reaction, we observed an increase in the apparent molecular weight of the TRAP cage after reaction with PTD4, as visualized by native PAGE (Figure 6c).

[0151] To be able to track the TRAP cage independently of its cargo, we labeled it with Alexa-647 fluorescent dye. For this, we crosslinked the maleimide groups on the dye with the 24 available cysteines lining the six 4-nm holes of the TRAP cage that are not involved in ring-ring interactions. By titration, we established the optimal amount of Alexa-647 (equal to the number of TRAP cysteine ​​groups) to be added, at which the TRAP cage was readily labeled and no free dye was present in the sample. This was assessed by native PAGE coupled with fluorescence measurements detecting both GFP(-21) and Alexa-647 (Fig. 6b). Although the cargo GFP contains three cysteine ​​residues, the control reaction showed no detectable labeling of GFP with Alexa647 (Fig. 6c). Negative staining transmission electron microscopy (TEM) confirmed that the modified TRAP cages retained their characteristic shape (Fig. 6d).

[0152] PTD4 peptide synthesis The PTD4 peptide derivative (Ac-YARAAARQARAG, (SEQ ID NO: 10), for brevity, referred to as PTD4 in the text) was synthesized on a 0.1 mmol scale using a Liberty Blue automated microwave synthesizer (CEM, USA) following Fmoc-based solid-phase peptide synthesis methodology. Fmoc-Gly-Wang resin (100-200 mesh, substitution 0.70 mmol / g, Novabiochem, Germany) was swollen overnight with dichloromethane (DCM) / dimethylformamide (DMF) (1:1). Fmoc deprotection was carried out with 25% morpholine in DMF at 85 °C for 5 min. Coupling reactions were carried out as per the recommended manufacturer's protocol using DIC / oxyma activator with a 5-fold excess of Fmoc-protected amino acid derivatives at 85 °C for 5 min. Double coupling was applied for all Fmoc-Arg(Pbf) couplings. N-terminal acetylation was performed on the resin with 10% acetic anhydride in DMF at 60°C. Cleavage from the resin and side chain deprotection were achieved by treatment with TFA / triisopropylsilane (TIS) / water (94:3:3) for 4 h with vigorous shaking at 30°C. The resin was filtered and the TFA was evaporated under a gentle stream of nitrogen. The crude peptide was precipitated by addition of cold diethyl ether followed by centrifugation (3000 rpm, 10 min). The resin was washed with cold ether (2x) and ethyl acetate (2x). The precipitated crude peptide was dried in vacuum overnight. The crude peptide was dissolved in 8 M urea and purified on an Agilent 1260 RP-HPLC using a semi-preparative C18 (10x150 mm) column (Cosmosil, Nacalai Tesque). The collected peptide-containing fractions were lyophilized. The purified peptides were analyzed on an analytical C18 column (Zorbax SBC18 5 mm 4.6 × 150 mm, Agilent) with a linear gradient of 0–20% acetonitrile with 0.1% TFA for 30 min at a flow rate of 1.0 ml / min. Peak signals were detected at 220 and 280 nm (Figure 6a).

[0153] TRAP cage labeling with Alexa-647 and decoration with cell-penetrating peptides Alexa Fluor-647 C2 maleimide fluorescent dye (Alexa-647, Thermo Fisher Scientific) and the cell-permeable PTD4 peptide were conjugated to the GFP-loaded TRAP cage via a cross-linking reaction with cysteines and lysines present in the TRAP protein.

[0154] To achieve fluorescent labeling, GFP-bearing TRAP cages (300 μl, 16 nM) were mixed with Alexa-647 C2 maleimide dye (50 μl, 1 μM) and the reaction was carried out in 50 mM HEPES with 150 mM NaCl pH 7.5 for 2.5 h at room temperature with continuous stirring at 450 rpm. The optimal interaction ratio of maleimide-conjugated Alexa-647 to TRAP cages was assessed by titration (Figure 6b). Briefly, aliquots of GFP(-21)-bearing TRAP cages (11.36 nM) were mixed with maleimide-conjugated Alexa-647 ranging from 0.1 μM to 100 μM. Samples were then separated by native gel electrophoresis and visualized by fluorescence detection in a Chemidoc with excitation at 647 nm. Reactions in which no free Alexa-647 was present in the samples and no GFP interference with the Alexa-647 signal was observed were considered as optimal decoration conditions and used in further experiments.

[0155] Additionally, to exclude the possibility of direct GFP labeling by Alexa-647, TRAP cages loaded with GFP(-21) with or without Alexa-647 labeling were subjected to denaturing gel separation and Western blotting followed by detection with anti-GFP antibody. No band shift due to potential interaction of GFP with Alexa-647 dye was observed (Fig. 6c).

[0156] For cell-penetrating peptide decoration, PTD4 peptide (50 μl, 0.5 mM) was mixed with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 10 μl, 83 mM) and N-hydroxysuccinimide (NHS, 10 μl, 435 mM), and all reagents were dissolved in ddH2O. Then, an excess of activated PTD4 peptide was added to the TRAP cage loaded with GFP(-21), labeled with Alexa-647, and incubated for the next 2.5 h at room temperature with continuous stirring at 450 rpm. The reaction was stopped by the addition of 5 μl of 200 mM Tris-HCl pH 7.5. Conjugation efficiency was verified by native PAGE and fluorescent gel imaging. The change in molecular weight of the decorated TRAP cage results in a band shift observed in native PAGE (Figure 6c).

[0157] Example 3. TRAP cage stability and effect on cell viability Before embarking on cell delivery testing, we first assessed whether the TRAP cage was structurally stable, i.e., would not disassemble under cell culture conditions. The stability was checked in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and 5% CO2 atmosphere with or without various concentrations of fetal bovine serum (FBS). The results showed that the cage structure was stable in DMEM culture medium within 18 hours of incubation at 37°C and 5% CO2 (Figure 7a).

[0158] To determine the effect of the TRAP cage on cell viability, the alamarBlue assay was performed. This test is based on the natural ability of viable cells to convert resazurin, a blue, non-fluorescent compound, to resofurin, a red, fluorescent molecule, by mitochondrial and other reductive enzymes. 9Human cancer cell lines MCF-7 and HeLa were incubated in the presence of TRAP cages, TRAP cages loaded with GFP(-21) and decorated with Alexa-647 and PTD4 peptides. The number of cells, TRAP cage dose, and stimulation time used in the cell viability test correspond to the conditions in which the TRAP cage internalization experiments were performed. Untreated cells were used as controls. The data showed that both the unmodified TRAP cages and the TRAP cages loaded with GFP(-21) and decorated with Alexa-647 and PTD4 did not significantly affect the viability of MCF-7 and HeLa cells for at least 4 hours of incubation (Figure 7b).

[0159] Cell culture and cytotoxicity assessment of TRAP cages: HeLa and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% FBS (EURx), 100 μg / ml streptomycin, 100 IU / ml penicillin (Gibco). Cultures were maintained at 37°C under 5% CO2. To test TRAP cage stability in culture medium, purified samples were added to DMEM medium containing 0, 2, and 10% fetal bovine serum (FBS) and incubated at 37°C under 5% CO2 for 2, 6, and 18 hours. Samples were then analyzed by native PAGE followed by Instant blue gel staining (Figure 7a).

[0160] Cell viability after TRAP cage treatment was determined using the alamarBlue test (VWR). Cells were cultured at 2.5 × 10 per well. 4The cells were cultured in 96-well plates at a density of 1 cell per well. The cells were then treated for 4 h with 5 μg (0.6 nM) of TRAP cages loaded with GFP (21) and decorated with Alexa-647 and PTD4 in 50 mM HEPES with 150 mM NaCl pH 7.5, supplemented with 10% FBS. After treatment, 10 μl of alamarBlue diluted in 90 μl DMEM medium was added per well and the cells were incubated for the next 3 h at 37 °C under 5% CO2. Resazurin, the active component of alamarBlue, is reduced to the highly fluorescent compound resofurin only in viable cells, and the absorbance of this dye (excitation 570 nm, emission 630 nm) was recorded. Untreated cells were used as a negative control (Figure 7b). All samples were measured in triplicate in three independent experiments.

[0161] Example 4. Delivery of protein cargo to cells The delivery of TRAP cages into cells was investigated using human cancer cell lines MCF-7 and HeLa. Cells were incubated with purified TRAP cages containing encapsulated GFP(-21) and labeled with Alexa-647 alone or with Alexa-647 and PTD4 for various periods of time and analyzed by flow cytometry. The fluorescent signals attributed to both Alexa647 and GFP increased with prolonged incubation times in both cell lines treated with GFP-bearing TRAP cages labeled with Alexa-647 and PTD4 peptides (Fig. 3a, b, c). These results indicate that external modification of TRAP cages with cell-penetrating peptides promotes their cell entry and effective cargo delivery. Interestingly, this effect was more pronounced in the case of MCF-7 cell line compared to HeLa cell line (Fig. 8a, b, c).

[0162] Confocal microscopy was used to distinguish the fluorescent signals from TRAP cages internalized in cells and those externally adsorbed at the cell membrane. TRAP cages containing GFP(-21) and labeled with Alexa-647 but lacking PTD4 were not observed in cells. In contrast, TRAP cages containing GFP(-21) and decorated with PTD4 showed obvious signals inside cells 4 hours after stimulation (Fig. 3d and Fig. 8d).

[0163] Example 5. Intracellular dynamics of the TRAP cage The high stability of the TRAP cage coupled with its ability to disassemble in the presence of moderate concentrations of cellular reducing agents suggests that the TRAP cage in the cytoplasm readily disassembles and releases the GFP(-21) cargo. Since the TRAP cage and GFP possess distinct and traceable signals, we hypothesized that cage disassembly and release of GFP(-21) may be strongly inferred if Alexa-647 and GFP signals are no longer colocalized after cell entry. To assess this possibility, we tracked both signals over time after addition to MCF-7 and HeLa cancer cells. Notably, in both tested cell lines, during the first 90 min of incubation, the TRAP cage was primarily present at the cell border, as indicated by the strong localization of the Alexa-647 signal there (Fig. 4a, 9a), and the GFP signal was almost undetectable (Fig. 4b, 9b). However, after 3 h of incubation, the TRAP cage signal (Alexa-647) became weaker and appeared to be more uniformly distributed within the cells, while the GFP signal was clearly detectable, likely due to its release from the TRAP cage (Fig. 4a, b and Fig. ​Fig.9a,b).

[0164] Example 6. Effect of Alexa-647 on GFP(-21) fluorescence To assess the potential effect of Alexa-647 on GFP(-21) fluorescence (suggested by Fig. 6a, middle panel), we compared cargo-loaded TRAP cages by confocal microscopy imaging, compared cages decorated with PTD4 peptide only or fully decorated (PTD4 and Alexa-647) (Fig. 10a). Briefly, cells were treated with each sample as described in Materials and Methods. Then, cells were fixed and stained according to the protocol described above. Fluorescence intensity in the green channel was quantified using ImageJ. Calculation of the mean fluorescence intensity (Fig. 10b) took into account the background signal from each field.

[0165] Additionally, the solution fluorescence of GFP(-21) encapsulated within a fully decorated TRAP cage was compared to the fluorescence of the cargo within the TRAP cage without Alexa-647 using an RF-6000 Shimadzu® spectrofluorometer. As shown in Figure 10c, the presence of Alexa-647 dye on the TRAP cage results in an approximately 30% decrease in the fluorescence of the cargo.

[0166] Example 7. Loading protein cargo into the TRAP cage by genetic fusion Efficient protein packaging was achieved by genetic fusion of guests to the cage-forming TRAP. As an initial model, we employed the far-red fluorescent protein mCherry (Fig. 11a). N-terminally His-tagged fluorescent proteins were fused to the TRAP, which faces inward when assembled. K35C The fusion proteins were genetically fused to the N-terminus of the unmodified TRAP in Escherichia coli host cells because excessive modification of a single 11-mer TRAP unit with these fluorescent proteins may prevent cage assembly due to steric hindrance. K35C and the individual transcript levels were increased by the differential inducers tetracycline and isopropyl-β- D-thiogalactoside (IPTG). Using this co-production system, the number of mCherry per TRAP ring can be fully regulated by changing the concentration of tetracycline. The patchwork TRAP ring fused to mCherry was then assembled into a cage using Au(I) (Figure 11b). Using the same approach, the TRAP cage can be loaded with more than one type of cargo protein.

[0167] Protein Production: To produce patchwork, TRAP rings were cotransformed with pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C. Protein expression was induced by the addition of 0.2 mM isopropyl-β-d-thiogalactopyranoside and tetracycline (8 ng / ml). After cell lysis by sonication, patchwork TRAP rings were then isolated using Ni-nitrilotriacetic acid (NTA) affinity chromatography followed by SEC using a Superdex 200 Increase 10 / 300 GL column.

[0168] Cage Assembly and Characterization: Formation of TRAP cages was performed by mixing equimolar amounts of purified TRAP rings containing mCherry and chloro(triphenylphosphinemonosulfoxide)gold(I)-(TPPMS-Au(I)-Cl) in 50 mM sodium phosphate buffer (pH 8.0) containing 600 mM NaCl and kept at room temperature overnight. Guest protein stoichiometry was determined using the absorbance ratio 280 / 587 nm. The morphology of isolated cages was examined using negative stain TEM using the protocol described above.

[0169] TRAP cage with mCherry decoration using cell-penetrating peptide: Maleimide moieties were introduced at the N-terminus of the peptides on the resin using 6-maleimidehexanoic acid and a DIC / Oxima coupling protocol. 0.5 mM 6-maleimidehexanoic-PTD4 or HA / E2 (25 μl, 0.5 mM) peptides were mixed with mCherry-loaded TRAP cages (75 μl, 0.3 mg / ml) and incubated overnight at room temperature with continuous stirring at 450 rpm. Conjugation efficiency was verified by native PAGE and fluorescent gel imaging. The change in molecular weight of the decorated TRAP cage results in a band shift observed in native PAGE.

[0170] Example 9. Loading the TRAP cage with two different protein cargoes by genetic fusion It is possible to load more than one type of protein into the TRAP cage. Two fluorescent proteins, mOrange2 and mCherry, which act as donor and acceptor of Förster resonance energy transfer (FRET), respectively, were encapsulated by genetic fusion of each cargo protein to the N-terminus of the TRAP monomer. The fusion proteins were co-produced with unmodified TRAPK35C in Escherichia coli host cells, and the individual transcription levels could be controlled by the different inducers tetracycline and isopropyl-β-D-thiogalactoside (IPTG). The amount of expression inducer added was optimized to obtain 0.3 mOrange2 and 1 mCherry protein per TRAP ring, which allows to avoid steric hindrance during the cage formation process.

[0171] The cargo-modified TRAP rings were then mixed in a 1:1 molar ratio and either Au(I)- or DTME was added to promote cage association (FIG. 12a).

[0172] The resulting cages were then purified by size-exclusion chromatography and analyzed by native PAGE coupled with fluorescence detection and TEM imaging (Figure 12b, c). Native PAGE confirmed successful encapsulation of the TRAP cage with both fluorescent cargoes, which could be viewed by fluorescence excitation at 532 nm (emission 610 nm) (Figure 12b).

[0173] TEM imaging showed a monodisperse population of distinctly cargo-packaged TRAP cages following their assembly with a mixture of cargo-modified TRAP rings, with the resulting ones retaining their morphology compared to the empty cages induced by Au(I) or DTME (Figure S1c).

[0174] The presence of mOrange2 and mCherry proteins in close proximity to the TRAP cage should enable Förster resonance energy transfer (FRET), a physical process in which energy is transferred from an excited fluorophore to another molecule. Energy transfer between fluorescent proteins encapsulated inside a protein cage has already been described, but has never been applied to monitoring the disassembly kinetics of an artificial protein cage.

[0175] TRAP Cage DTME and TRAP Cage Au(I) To assess the efficiency of FRET between mOrange2 and mCherry proteins inside the TRAP cage, spectral data were collected using excitation values ​​relative to mOrange2. All spectral data were normalized to the mOrange2 fluorescence peak, and colocalization with mCherry was determined by the relative fluorescence intensity ratio. Fluorescence spectra were collected for TRAP cages that encapsulated either mCherry or mOrange2 proteins alone, as well as for TRAP cages packaged with a FRET pair. Au(I) / DTMEThe measurements were also performed against a control sample in which the fluorophores were encapsulated in separate cages and subsequently mixed in solution. Such a control sample would not show FRET because the fluorophores are far from each other. In fact, the FRET pair was encapsulated in a TRAP cage packaged Au(I) and TRAP Cage DTME The spectra of mOrange2 and mCherry showed approximately 1.5-fold higher signal of mCherry emission at 610 nm compared to the corresponding control sample (Fig. S13a, b), indicating efficient energy transfer between mOrange2 and mCherry inside both DTME- and Au(I)-induced TRAP cages.

[0176] Protein Production: E. coli strain BL21(DE3) cells were co-transformed with either pACTet_H-mOrange-TRAPK35C or pACTet_H-mCherry-TRAP-K35C and pET21_TRAP-K35C. Cells were grown at OD 600=0.5-0.7. At this point, protein expression was induced by the addition of 0.2 mM IPTG and 10 ng / ml tetracycline in the case of pACTet_H-mCherry-TRAP-K35C or 30 ng / ml tetracycline in the case of pACTet_H-mOrange-TRAP-K35C, followed by 20 h incubation at 25 °C. Cells were then harvested by centrifugation at 5,000 × g for 10 min. Cell pellets were stored at -80 °C until purification. The pellet was resuspended in 40 ml lysis buffer (50 mM sodium phosphate buffer, 600 mM NaCl, 10 mM imidazole, pH 7.4) supplemented with DNase I and lysozyme, 1 tablet of protease inhibitor cocktail, and 2 mM DTT, and stirred at room temperature for 30 min. The samples were then sonicated and clarified by centrifugation at 10,000×g for 20 min at 4° C. The supernatant was then incubated with 4 ml Ni-NTA resin pre-equilibrated in lysis buffer in a gravity-flow column for 20 min. The resin was then washed over 10 column volumes in lysis buffer containing 20 and 40 mM imidazole. His-tagged proteins were eluted using 5 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 500 mM imidazole. The protein samples were then buffer exchanged into 2× phosphate buffered saline (PBS) + 5 mM ethylenediaminetetraacetic acid (EDTA), hereafter referred to as 2× PBS-E, using Amicon Ultra-15 centrifugal filter units (50k molecular weight cut-off (MWCO), Merck Millipore). The protein was then subjected to size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.8 ml / min. The major peaks absorbing at 548 nm or 587 nm were pooled and concentrated using an Amicon Ultra-15 (50k MWCO). Protein purity was checked by SDD-PAGE and protein concentration was determined by the extinction coefficient: ε mCherry 587 =72,000M -1 cm -1, ε mOrange 548 =58,000M -1 cm -1 , ε TRAP 280 =8250M -1 cm -1 The protein was determined by absorbance measured using a UV-1900 UV-Vis spectrophotometer (Shimadzu Corporation) using the ELISA kit (http: / / expasy.org / tools / protparam.html). The protein was stored at 4°C until use.

[0177] Cage Assembly and Purification: For crosslinker-induced cage association, TRAP(K35C / R64S) (100–500 μM) in 2× PBS-E was mixed with a 5-fold molar excess of either DTME or BMH and stirred at room temperature for 1 h. The final DMSO concentration in the solution was kept no greater than 12.5%. After the reaction, the insoluble fraction, which was probably due to the low solubility of the crosslinker in aqueous solution, was removed by centrifugation at 12,000×g for 5 min. The supernatant was then purified by size-exclusion chromatography using a Superose 6 Increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.5 ml / min on an AKTA purifier FPLC (GE Healthcare). The fractions containing crosslinked TRAP cages were pooled and concentrated using an Amicon Ultra-4 (100k MWCO) centrifugal filter unit. The typical yield of crosslinked TRAP cages obtained was approximately 20%. Formation and purification of gold(I)-induced TRAP cages were performed as described previously (1). Cage formation with fusion proteins was performed using the same protocol as described for both cross-linked and gold(I)-induced cages, with an additional Ni-NTA purification step before size-exclusion chromatography to purify samples from partially assembled cages (His-tagged mCherry and mOrange2, which are not fully protected inside the cage, bind to a Ni-NTA column). Protein concentrations and ratios of encapsulated guests were estimated using absorbance ratios at 280 / 548 nm or 280 / 587 nm, using a method similar to that previously reported (4). The extinction coefficient used in the calculation was ε mCherry 587 =72000M -1 cm -1 , ε mOrange2 548 =58000M -1 cm -1 , ε TRAP 280 =8250M -1 cm -1Due to the spectral overlap between mCherry and mOrange2, to properly calculate the concentrations of both encapsulated guests, the mCherry extinction coefficient was also estimated at 548 nm (ε mCherry 548 =42538M -1 cm -1 Similarly, the extinction coefficients of mCherry and mOrange2 at 280 nm are ε mCherry 280 =56744M -1 cm -1 and ε mOrange2 280 =52200M -1 cm -1 The morphological fidelity of the assembled cages was confirmed by negative staining TEM and native PAGE analysis.

[0178] Fluorescence measurements: Fluorescence spectra were acquired at room temperature on a Shimadzu RF-6000 fluorescence spectrophotometer using 70 nM mOrange2 in 2x PBS-E in 1-cm pathlength polystyrene cuvettes. Proteins were excited at 510 nm and emission was scanned over the wavelength range of 530-700 nm. Acquired spectra were normalized to the mOrange2 fluorescence peak. After each measurement, 10 mM DTT was added to the samples to induce complete uncaging.

[0179] [Table 4]

[0180] Example 10. Loading protein cargo into the TRAP cage via isopeptide bond formation Despite the robust and general system using genetic fusion, this strategy still suffers from the requirement of exposing the guest protein to Au(I) or maleimide crosslinkers. This procedure can be particularly problematic when the guest protein contains a free cysteine ​​residue that is important for activity, for example in the case of cysteine ​​proteases. To overcome the problem, we devised a post-association loading system using the SpyTag / SpyCatcher system, where the 13 amino acid peptide SpyTag interacts with the protein SpyCatcher to spontaneously form an isopeptide bond (Zakeri B et al., Proc. Natl. Acad. Sci. USA 2012, 109, 690-697, hereby incorporated by reference). In the context of loading into protein cages, two strategies can be used (Figure 14a). In strategy 1, the inner wall of the TRAP cage carries SpyCatcher, while the cargo carries SpyTag. In the second strategy, the inner wall of the TRAP cage carries SpyTag, while the cargo carries SpyCatcher. We first designed various constructs based on this approach for the capture of GFP in the TRAP cage (Figure 14b). For strategy 2, SpyCatcher was introduced either at the N-terminus of TRAP or in the lumen-facing loop (specifically between 47 and 48). All host TRAPs are equipped with a hexahistidine tag to facilitate purification, while the tag can be cleaved off using either carboxypeptidase or SUMO protease to yield TRAP-K35C variants possessing SpyTag or SpyCatcher at the N-terminus, respectively, designated SpyT-TRAP or SpyC-TRAP, as well as one with SpyCatcher in the lumen loop, designated TRAP-loopSpyC.

[0181] SpyCatcher-bearing TRAP variants were co-produced in host bacteria with untagged TRAP-K35C as described in Example 9 to produce patchwork rings. To avoid insufficient cage formation due to excessive SpyCatcher moieties, we tested two concentrations of tetracycline, 10 or 30 ng / mL, to regulate the gene expression level of SpyCatcher-fusion variants. The production of patchwork rings with varying contents of fusions as well as the cleavage of His-tag by SUMO protease was confirmed by SDS-PAGE analysis (Fig. 15a). With the addition of Au(I) in phosphate buffer, all variants showed cage formation, while the association efficiency was further enhanced by increasing the ionic strength of the buffer (Fig. 15b). After isolation of the assembled cages, they were mixed with varying concentrations of GFP with SpyTag (SpyTag-GFP) in phosphate-buffered saline (PBS). Isopeptide formation as well as host-guest association, as judged by analysis using SDS-PAGE (Fig. S16a) and native PAGE (Fig. S16b), were observed for all variants, indicating successful guest packaging. However, further analysis of SpyC-TRAP cage samples acquired from 30 ng / mL tetracycline using SEC and TEM showed a slight shift in elution time compared to the empty TRAP cage, and some objects outside the cage assembly, suggesting that guest GFP may partially leak out of the cage (Fig. S17). On the other hand, no such leakage was observed for the TRAP-loop SpyC cage. UV-Vis absorbance of isolated filled cages revealed that these TRAP cages could carry up to about 28 GFP molecules, while the packaging density could be controlled by varying either the tetracycline concentration in the bacterial production or the mixing ratio in the encapsulation process. In contrast to the TRAP-SpyCatcher fusion, strategy 1 proved challenging as the SpyT-TRAP variant exhibited a tendency towards aggregation and therefore efficient Au(I)-mediated cage formation was not observed (Figure 17c).Taken all together, it can be concluded that the TRAP-loop SpyC variant is the most robust and efficient variant for guest packaging.

[0182] The second guest demonstrated in this manner was SpyTagged Neoleukin-2 / 15 (Silva DA et al., Nature, 2019, 565, 186-191, hereby incorporated by reference), which was also successfully loaded into a TRAP cage possessing a SpyCatcher in the lumen. The light-gated TRAP was composed of a variant containing two mutations, K35C and R64S, and lacking the two C-terminal lysine residues, d73K and d74K, and a TRAP-loop SpyC, with the TRAP rings connected to each other using the photocleavable crosslinker 1,2-bisbromomethyl-3-nitrobenzene (BBN) (Figure 18). As in the case of GFP, SpyTagged NL-2 was mixed with the TRAP cage, and the loaded cage was isolated by size-exclusion chromatography (Figure 19a). Subsequent analysis using negative stain TEM and SDS-PAGE revealed that the NL-2 protein was successfully packaged into TRAP via isopeptide bond formation (FIG. 19b).

[0183] The properties of SpyTag-NL-2 encapsulated within the TRAP cage were assessed using the HEK-Blue IL-2 cell assay. HEK-Blue is a type of HEK293T cell engineered to stably co-express the human IL-2 receptor along with its signaling pathway and an additional secreted embryonic alkaline phosphatase (SEAP) reporter gene. Binding of IL-2 or NL-2 to the IL-2 receptor (IL-2R) leads to the initiation of a signaling cascade that results in transcriptional activation and secretion of SEAP, allowing its monitoring by colorimetric methods.

[0184] HEK-Blue cells were seeded in 96-well plates. The next day, NL-2 / 15 encapsulated and empty UV-photocleavable SpyCatcher-TRAP cage samples were supplemented with 10 mM cysteine ​​(quencher) and treated with UV light for 10 min. Treated samples and controls were diluted in cell culture medium (DMEM) to various concentrations in the pM range. Control samples included unconjugated SpyTag-NL-2 and purchased human IL-2, SpyTag-NL-2 conjugated with TRAP rings, and empty TRAP cages, before and after UV treatment. Cells were stimulated for 24 h, followed by a Quanti Blue assay that allows to assess the amount of secreted SEAP.

[0185] HEK-Blue cells treated with SpyTag-NL-2, hIL-2, and TRAP-NL-2 control samples showed very similar levels of produced SEAP after stimulation, suggesting that IL-2R binding is not affected by conjugation of NL-2 / 15 to the TRAP ring and its modification with SpyTag (Figure 20a). Treatment of cells with empty TRAP cages did not result in any signal transduction before or after UV irradiation of the samples (Figure 20b).

[0186] The production of SEAP was prominent after treatment with NL-2 loaded TRAP cages after UV irradiation (Figure 20b). Samples without UV light treatment were also capable of signal transduction through IL-2R but at a much lower level.

[0187] Protein Production: Patchwork constructs composed of TRAP variants containing the K35C mutation, N-terminal His6-SUMO, and SpyCatcher either downstream of SUMO or between residues 47 and 48, along with TRAP-K35C (or, for NL-2 encapsulation, TRAP-K35C, R64S, d73K, d74K), were produced using essentially the same protocol as for the mCherry fusion. Tetracycline (10 or 30 ng / mL) and ITPG (0.2 mM) were used to induce protein expression. After cell lysis by sonication, the fusion protein was purified from the soluble fraction using Ni-NTA affinity chromatography. The His6-SUMO unit was then cleaved from the full-length fusion by overnight treatment with SUMO protease 1 (25 units / mg of total protein) at 4°C, followed by treatment with Ni-NTA agarose resin to remove unreacted species and his-tagged proteases. The desired patchwork assembly was further purified by size-exclusion chromatography. The fidelity of the protein as well as the number of SpyCatchers per 11-mer TRAP ring were estimated using the band intensity ratio in SDS-PAGE analysis.

[0188] N-terminal His6 and SpyTagged GFP and neoleukin-2 / 15, designated H-SpyT-GFP or H-SpyT-NL-2, were produced using E. coli BL21(DE3) strain transformed with pET28_H-SpyT-GFP or pET28_H-SpyT-NL-2, pET28-based plasmids carrying ColE origin of replication, kanamycin resistance gene, lac repressor, and H-SpyT-GFP or H-SpyT-NL-2 under the control of T7 promoter and lac operon system. Proteins were expressed for 20 h at 25°C using 0.2 mM IPTG and purified using Ni-NTA affinity chromatography and size exclusion chromatography.

[0189] Cage assembly and characterization: Patchwork TRAP rings containing SpyCatcher (400 μM for TRAP monomers) were mixed with TPPMS-Au(I)-Cl (200 μM) in 50 mM sodium phosphate buffer (pH 8.0) containing 600 mM NaCl (2 M NaCl for those containing TRAP-K35C, R64S, d73K, d74K) and kept at room temperature overnight. The assembled cages were then isolated using size-exclusion chromatography. For the photo-openable cages, 1,2-bromomethyl-3-nitrobenzene (300 μM, 3 equivalents (euiv.)) in DMF (5% final) was added to the Au(I)-mediated cages (200 μM for TRAP monomers) and stirred at room temperature for 1 h. β-mercaptoethanol (4 μL) was then added to the reaction and further stirred at room temperature for 30 min to quench unreacted benzyl bromide and remove Au(I). These small molecule reactants were removed by ultrafiltration using an Amicon ultra-4 centrifugal unit (30,000 molecular weight cutoff) and the resulting cages were used for encapsulation without further purification. The number of SpyCatchers per cage was estimated using the band intensity ratio in SDS-PAGE analysis. The morphology of the isolated cages was examined using negative stain TEM using the protocol described above.

[0190] Guest loading (small scale): Patchwork TRAP rings containing SpyCatcher (20 μM, for SpyCatcher) were mixed with H-SpyT-GFP (0-20 μM) in PBS and kept at room temperature overnight. The reaction mixture was then analyzed by SDS-PAGE and native PAGE. For native PAGE analysis, bands were visualized using both Instant Blue staining and fluorescence using blue light excitation and emission filters (530 / 28 nm) on a Biorad ChemiDoc MP imager.

[0191] Guest loading (large scale): Patchwork TRAP rings containing SpyCatcher (20 μM, for SpyCatcher) were mixed with H-SpyT-GFP (20 μM) or H-SpyT-NL-2 (40 μM) in PBS and kept at room temperature overnight. Cages were then isolated by size-exclusion chromatography using a Superose 6 increase 10 / 300 column, followed by TEM and spectroscopic analysis. TEM imaging was performed as described above. The number of guests per cage was determined by the extinction coefficient: ε GFP 488 =52,700M -1 cm -1 , ε GFP 280 =26,850M -1 cm -1 , ε TRAP 280 =8250M -1 cm -1 The concentration was estimated by absorbance measured with a UV-1900 UV-Vis spectrophotometer (Shimadzu Corporation) using the FTIR (http: / / expasy.org / tools / protparam.html).

[0192] [Table 5-1] [Table 5-2]

[0193] HEK-Blue-IL-2 reporter cell assay HEK-Blue-IL-2 cells were cultured in DMEM-high glucose medium with 10% FBS, 100U / mL penicillin, 100ug / mL streptomycin, and 50ug / mL Normocin. After passage 2, cells were also supplemented with HEK-Blue™ CLR Selection and puromycin to ensure sustained transgene expression in the cells. Prior to seeding, the CLR selection medium was replaced with DMEM-high glucose medium with 10% FBS, 100U / mL penicillin, 100ug / mL streptomycin (P / S), and 50ug / mL normocin. Cells were detached from the surface of the culture flask (VWR) by a thin stream, centrifuged at 70×g for 8 minutes, and resuspended in 2 ml of fresh medium without normocin addition. To assess their numbers, 10 μl of the cell suspension was transferred to a counting plate (BioRad) and placed in a TC20 automated cell counter (BioRad). 4 The cells were seeded at a density of 100x in 180 ul culture medium into 96-well culture plates (VWR) and incubated at 37° C. and 5% CO 2 for 2 hours.

[0194] Tested proteins were prepared as 10x stock dilutions in DMEM-high glucose with 10% FBS. The next day, HEK-Blue-IL-2 cells were stimulated by the addition of 20μl of proteins at various concentrations and incubated for 24 hours at 37°C and 5% CO2. Quanti-Blue™ solution was prepared by 100x dilution of QB-buffer and QB-Reagent in sterile H2O and incubated for 10 minutes with gentle shaking, protected from light. 180ul of Quanti-Blue™ solution was transferred to each well of a new 96-well plate and 20μl of HEK-Blue-IL-2 cell supernatant was added. The plate was incubated for 1 hour at 37°C. Secreted embryonic alkaline phosphatase (SEAP) activity was assessed by absorbance measurement at 630nm.

[0195] References 1 Malay、A. D. et al. An ultra-stable gold-coordinated protein cage displaying reversible assembly. Nature 569、438-442 (2019). 2 Butterfield、G. L. et al. Evolution of a designed protein assembly encapsulating its own RNA genome. Nature 552、415-420 (2017). 3 Edwardson、T. G.、Mori、T. & Hilvert、D. Rational Engineering of a Designed Protein Cage for siRNA Delivery. J. Am. Chem. Soc. (2018). 4 Azuma、Y.、Zschoche、R.、Tinzl、M. & Hilvert、D. Quantitative packaging of active enzymes into a protein cage. Angew. Chem. Int. Ed. 55、1531-1534 (2016). 5 Dashti、N. H.、Abidin、R. S. & Sainsbury、F. Programmable in vitro coencapsidation of guest proteins for intracellular delivery by virus-like particles. ACS nano 12、4615-4623 (2018). 6 Woersdoerfer、B.、Pianowski、Z. & Hilvert、D. Efficient in vitro encapsulation of protein cargo by an engineered protein container. Journal of the American Chemical Society 134、909-911 (2012). 7 Ho、A.、Schwarze、S. R.、Mermelstein、S. J.、Waksman、G. & Dowdy、S. F. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer research 61、474-477 (2001). 8 Berry、C. C. Intracellular delivery of nanoparticles via the HIV-1 tat protein. Nanomedicine 3、357 - 365 (2008). 9 Rampersad、S. N. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors 12、12347-12360 (2012).

Claims

1. An artificial TRAP cage comprising a selected number of TRAP rings and at least one guest cargo encapsulated therein, wherein the guest cargo is selected from the group comprising proteins, enzymes, antigens, antibodies, protein macromolecules, lipids, peptides, nucleic acids, small molecule cargo, peptide nucleic acids, carbon-based structures, metals, toxins, or nanoparticles.

2. The nucleic acid is selected from the group comprising DNA, RNA, mRNA, siRNA, tRNA, and microRNA; the enzyme is an enzyme associated with overexpression in a metabolic disorder or disease, or underexpression in a metabolic disorder or disease, optionally the enzyme is selected from the group comprising hydrogenases, dehydrogenases, lipases, lyases, ligases, proteases, transferases, reductases, recombinases, and nucleases acid-modifying enzymes; the therapeutic agent is selected from the group including cancer therapeutics, anti-infective therapeutics, vascular disease therapeutics, immunotherapeutics, senolytics, and neurological therapeutics; the metal is selected from the group including iron, zinc, platinum, copper, sodium, cadmium, lanthanides, gadolinium, technetium, calcium, potassium, chromium, magnesium, molybdenum, and salts or complexes thereof; The toxin is selected from the group including ligand-targeted toxins, protease-activated toxins, melittin, and toxin-based suicide gene therapy drugs. The guest cargo is a protein, preferably the protein is a fluorescent protein, interleukin-2 (IL-2), or neoleukin-2 / 15 (NL-2); 2. The cage of claim 1.

3. 3. The cage of claim 1 or 2, wherein the cage comprises a plurality of guest cargoes, the guest cargoes being the same or different from one another.

4. Further comprising at least one external decoration; Optionally, at least one of the external decorations comprises a cell permeation agent that facilitates intracellular delivery of the cage containing the internal guest cargo; Further optionally, the cell penetrating agent is PTD4. A cage according to any one of claims 1 to 3.

5. The number of TRAP rings in the TRAP cage is 6 to 60, Optionally, the number of TRAP rings in the TRAP cage is 12, 20 or 24, preferably 24; A cage according to any one of claims 1 to 4.

6. 6. The cage of claim 1 , wherein the inner surface of the TRAP cage lumen is highly charged and the TRAP cage protein comprises an E48Q or E48K mutation.

7. 7. The cage of any one of claims 1 to 6, wherein the artificial TRAP cage protein is modified to comprise any one or more of the mutations selected from the group comprising: K35C, K35H, R64S, E48Q, E48K, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, S33H / K35C, K35C / E48Q, K35C / E48K, K35H / E48Q, K35H / E48K, S33C / E48Q, S33C / E48K, S33C / E48Q, and S33C / E48K.

8. The cage opening is programmable, Optionally, the programmable opening of the cage relies on the selection of a molecular or atomic crosslinker that holds the TRAP ring in place in the TRAP cage; Further optionally, the crosslinker is either (i) a reduction-responsive / sensitive linker, such that the cage opens under reducing conditions; or (ii) a photoactivatable linker, such that the cage opens upon exposure to light. A TRAP cage according to any one of claims 1 to 7.

9. The TRAP cage according to any one of claims 1 to 8, for use as a delivery vehicle for intracellular delivery of its internal guest cargo, for use as a vaccine, for use as a medicine, or for the treatment of a disease or condition selected from the group including cancer, vascular disease, cardiovascular disease, diabetes, infectious disease, autoimmune conditions, neurodegenerative diseases, cellular aging diseases, arthritis, and respiratory diseases.

10. (i) obtaining a TRAP ring unit by expression of the TRAP ring unit in a suitable expression system and purification of said unit from the expression system; (ii) conjugation of a TRAP ring unit via at least one free thiol linkage with a crosslinker; (iii) modification of the TRAP ring unit to provide a suitable internal environment for trapping guest cargo; (iv) self-assembly to form a TRAP cage to provide a cage lumen in which guest cargo is encapsulated; and (v) Purification and isolation of TRAP cages encapsulating guest cargo 1. A method for making an artificial TRAP cage with encapsulated guest cargo comprising: The guest cargo can be loaded either before or after assembly of the TRAP cage; The method, wherein the guest cargo is selected from the group comprising a protein, an enzyme, an antigen, an antibody, a protein macromolecule, a lipid, a peptide, a nucleic acid, a small molecule cargo, a peptide nucleic acid, a carbon-based structure, a metal, a toxin, or a nanoparticle.

11. The modification in step (iii) is (i) highly charging the inner surface of the TRAP cage lumen; (ii) genetic fusion of guest cargo to the inner surface of the TRAP cage lumen; (iii) SpyCatcher / SpyTag conjugation of guest cargo to the inner surface of the TRAP cage lumen; and (iv) via covalent bond formation in both chemical and enzymatic methods The method of claim 10, wherein the compound is selected from the group comprising:

12. Step (ii) comprises firstly the conjugation of the TRAP ring units via at least one metal crosslinker, preferably an atomic metal crosslinker, followed by a step of replacing the metal crosslinker with a molecular crosslinker; and / or The highly charging of the inner surface of step (i) provides either a net positive or a net negative charge on the inner surface of the cage lumen; 12. The method according to claim 10 or 11.

13. the artificial TRAP cage protein is modified to include any one or more of the mutations selected from the group comprising: K35C, K35H, R64S, E48Q, E48K, K35C / R64S, K35H / R64S, S33C, S33H, S33C / R64S, S33H / R64S, S33C / K35H, S33H / K35H, S33C / K35C, S33H / K35C, K35C / E48Q, K35C / E48K, K35H / E48Q, K35H / E48K, S33C / E48Q, S33C / E48K, S33C / E48Q, and S33C / E48K; Optionally, TRAP Ｋ３５Ｃ Ｅ４８Ｑ is carried out in a sodium bicarbonate buffer at pH 9-11; or Optionally, TRAP Ｋ３５Ｃ Ｅ４８ｋ is carried out in sodium bicarbonate buffer at pH 10-10.5; The method according to any one of claims 10 to 12.

14. The genetic fusion of guest cargo to the inner surface of the TRAP cage lumen in step (ii) results in TRAP Ｋ３５Ｃ and / or optionally, in the SpyCatcher / SpyTag conjugation of the guest cargo to the inner surface of the TRAP cage lumen in step (iii), SpyCatcher is introduced into the loop region of the TRAP ring between residues 47 and 48 that faces inward when associated with the TRAP cage, and the guest cargo contains a SpyTag; and / or The enzymatic modification is mediated by a peptide ligase selected from the group including sortase, asparaginyl endoprotease, trypsin-related enzymes, and subtilisin-derived variants, and the covalent chemical bond formation can include strain-promoted alkyne-azide cycloaddition and pseudopeptide bond formation.

14. The method according to any one of claims 11 to 13.

15. A TRAP cage produced by the method of any one of claims 11 to 14.

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